
Class 

Book 

()qpyiig]it>]°_ 



Ci)K»:lGRT DEPOSm 



SOIL BIOLOGY 



LABORATORY MANUAL 



BJ 



ALBERT LEMUEL WHITING, Ph.D., 

Associate in Soil Biology in University of Illinois, 

College of Agriculture and Agricultural 

Experiment Station 



FIBST EDITION 



NEW YORK 

JOHN WILEY & SONS, Inc. 

London: CHAPMAN & HALL, Limited 

1917 



>/- 






Copyright, 1917, 

BY 

ALBERT LEMUEL WHITING 



MAR 20 1917- 



Stanbope jprcss 

F. H. GILSON COMPANY 
BOSTON, U.S.A. 



ICI.A455963 



PREFACE. 



Soil biology treats of the microorganisms which inhabit 
soils in their relation to soil fertiUty, crop production, and 
permanent agriculture. It includes the rapidly develop- 
ing fields of soil bacteriology, soil protozoology, soil 
mycology, and others which may later merit study. The 
purpose of this manual is to present the important prin- 
ciples of soil biology, particularly as they point to the 
inteUigent control of the essential elements of plant food. 
The principles are incorporated in practices which acquaint 
the student with the various forms of life in the soil and 
their activity. Special attention is given to all biochemi- 
cal reactions influencing soil conditions. The sequence of 
arrangement of the practices is not fixed but that given 
has been found best in this laboratory. The choice of 
materials which are tested is based upon farm practice. 
Students are encouraged to undertake these studies on 
their own soils. 

^The laboratory course is a part of a five-hour course 
consisting of two lectures, one quiz, and three laboratory 
periods per week. 

Soil fertility and bacteriology are prerequisites while 
organic chemistry and plant physiology are desirable for 
the course as outHned. 

The questions, problems, and references accompanying 
the practices have been found by experience to be valuable 
supplements in fixing the principles and applying the 
information obtained. 

Emphasis is laid upon quantitative results and the 
measure appHed consists of biochemical and chemical 
methods. The results thus obtained are interpreted as 
far as possible in terms of soil.fertility and crop yields. 



iv PREFACE 

In Part II are included bacteriological, chemical, me- 
chanical, and pot-culture methods as appHed or developed 
in this laboratory. In the last section will be found 
Suggestions for Instructors and Students Preparing to 
Teach. An attempt has been made to satisfy the demand 
to have this information ever at hand and in a classified 
form. 

This little work would not be complete without an 
acknowledgment to Professor C. G. Hopkins for sugges- 
tions and encouragement in its development and to Mr. 
Warren R. Schoonover, assistant in soil biology, whose 
able assistance and careful observations have proved 
invaluable; also to certain former graduate students for 
testing new methods. 

A. L. WHITING. 

Urbana, Illinois, 
February, 1917. 



TABLE OF CONTENTS. 



PART I pagb 

Examination of Microorganisms in Soils and Manures 3 

Occurrence of Bacteria at Different Depths in Soils 6 

Quantitative Bacteriological Examination of Soils 6 

Occmrence of Non-vegetative Forms of Bacteria and Fungi 

in Soils 10 

Occurrence of Fungi at Different Depths and in Different 

SoUs 12 

Ammonification in Soils 14 

Influence of Moisture Content on the Process 14 

Ammonification of Urea and Isolation of Urea Organisms. . . 17 

Nitritation 20 

Oxidation of Ammonia to Nitrite by Nitrosomonas .... 20 
Influence Exerted by Carbonates, Ignited Soil, Soluble 

Organic Matter and Aeration 20 

Nitratation 23 

Oxidation of Nitrite to Nitrate by Nitrobacter 23 

Influence Exerted by Carbonates, Ignited Soil, Soluble 

Organic Matter and Aeration 23 

Isolation and Study in Pure Culture of Nitrosomonas and 

Nitrobacter 26 

Production of Inorganic Nitrogen in Soils 28 

Comparative Ammonification and Nitration of Crop 
Residues, Green Manures, and Farm Manures 

' in Typical Soils 28 

Influence Exerted by Carbonates, Soil Type, Moisture 

and the Conditions of the Organic Substances 28 

Carbon Dioxid Production 36 

CeUiilose Decomposition ....'. 39 

Aerobic Decomposition by Fungi and Bacteria 39 

Cellulose Decomposition 41 

Anaerobic Decomposition 41 

Symbiotic Nitrogen Fixation 43 

Inoculation of Legume Seeds 43 

Growth of Nodules and Determination of Nitrogen Fixed 43 

V 



Vi TABLE OF CONTENTS 

Page 

Isolation of B. Radicicola from Legume Nodules 46 

Non-Symbiotic Nitrogen Fixation 48 

Aerobic Nitrogen Fixation in Soils ....*..... 48 

Influence of Carbonates on the Process 48 

Nitrogen Fixation in Solution by Soil Bacteria 51 

Isolation of Azotobacter from Soil 53 

Non-symbiotic Anaerobic Nitrogen Fixation and Isolation of 

B. Clostridium Pasteurianum 55 

Denitrification and Formation of Calcium Carbonate 57 

Sulfofication and Desulfofication in Soils 60 

Fungi in Soils 63 

Relation to Soil Nitrogen 63 

Factors Influencing Fungous Growths in Soils 63 

Protozoa in Soils 65 

Isolation and Study of Amoebse, Ciliates and Flagellates 

from Soil 65 

Determination of Active Protozoa in Soils 65 

Algse in Soils 68 

Relation to Soil Nitrogen 68 

Comparative Numbers in Different Soils 68 

Factors Influencing the Growth of Algse in Soils 68 

A Study of Enzymes 71 

Growth and Study of Iron Bacteria of Soils 73 

Denitrification in Solution by Soil Bacteria 75 

Active Protozoa in Soils 76 

Decomposition of Cyanamid 78 

Protein Formation in Soils 81 

Flagella Staining of B. Radicicola, B. Nitrosomonas, 

B. Denitrificans, B. SubtUis, B. Typhosus .... 83 

Cross Inoculation of Legumes 84 

Solvent Action of Soil Bacteria on Minerals 86 

Soluble Phosphorus and Calcium Produced by Nitrosomonas 86 

PART II 

Bacteriological Methods 91 

Food of Soil Microorganisms 91 

Preparation of Culture Media 93 

Formulae of Solutions (Liquid Media) 95 

Formulae of Solid Culture Media 97 

Special Media 99 

SHica Jelly 99 

Magnesium Plaster of Paris Blocks 100 



TABLE OF CONTENTS VU 

Page 

Cellulose Solution 100 

Reaction of Culture Media 101 

Staining and Preparation of Stains 103 

Simple Staining 103 

Special Staining 103 

Flagella Stains (Loeffler's) 104 

Spore Stain (Hansen's) 104 

Capsule Stain (Hiss') 105 

Nodule Tissue Stain (Flemming's) 105 

Protozoa Fixative Stain and Mount (Martin and 

Lewin .• 105 

JProtozoa Stain 106 

Algse Fixative and Stain 107 

Formulae of Stains 107 

Formulae of Stock Solutions of Simple Stains 107 

Formulae of Simple Stains 108 

Formulae of Stock Solutions of Disinfectants 109 

Formulae of Special Stains 109 

Fixatives 110 

Chemical Methods Ill 

Quantitative Determination of Nitrogen 108 

Total Nitrogen in Soil Ill 

Total Organic Nitrogen in Soil 112 

Total Nitrogen in Microorganisms, Plants and Other 

Organic Materials 112 

Ammonia Nitrogen 112 

Ammonia Nitrogen by Aeration 113 

Nitrite Nitrogen 113 

Nitrate Nitrogen 113 

Nitrite and Nitrate Nitrogen 114 

Inorganic Nitrogen 114 

Qualitative Tests for Nitrogen 115 

Organic Nitrogen . 115 

Test for Organic Nitrogen and Sulfur when Present 

Together 116 

Ammonia 116 

Nitrites -. 116 

Nitrates 117 

Quantitative Determination of Sulfur 117 

Total Sulfur in Soil and Organic Materials 117 

Sulfates in Soil 118 

Qualitative Test for Sulfur 119 

Organic SuKur 119 



VIU TABLE OF CONTENTS 

Page 

Inorganic Sulfate 119 

Hydrogen Sulfid 119 

Determination of Phosphorus, Carbon, Dry Matter, 

Acidity and Magnesium 119 

Determination of Calcium 119 

Determination of Iron (Total) 119 

Determination of Carbon Dioxid 120 

Mechanical Methods 121 

Collecting Soil Samples for Biochemical Analysis . . 121 

Collecting SoU Samples for Bacteriological Analysis 121 

Preparation of Soil Samples 122 

Sampling of Crops 123 

Shaking 123 

Preparing a Soil Infusion 123 

Ignition of Soil 123 

Centrifuge ". . 123 

Filtration 124 

Cleaning Glassware 124 

Autoclave 125 

Hot Air Oven 126 

Sterilization of Glassware 126 

Sterilization of Seeds 127 

Sterilization of Nodules 128 

Sterilization of Parts of Plants 128 

Sterilization of Soil 128 

Moist Heat 129 

. Dry Heat 129 

Volatile Antiseptics 131 

Sterilization of Sand 131 

Pot Culture Methods 132 

Containers 132 

Sand Medium 132 

Soil Medium 132 

Moisture 132 

Plant Food 133 

Inoculation of Legume Seeds 133 

Planting 133 

Crops 134 

Growth of Plants Under Sterile Conditions 134 

Records 134 

Suggestions for Instructors and Students Preparing to Teach 136 

Acid, Alkah and Other Standard Solutions 136 

Indicators 137 



TABLE OF CONTENTS IX 

Page 

Colorometric Reagents 138 

Chemicals Used by Students in Soil Biology 139 

Apparatus 141 

Special Apparatus 142 



PART I. 
LABORATORY PRACTICES. 

1. Pkactices 1-25 

2. Class Practices 1-2 

3. Advanced Practices. . 1-6 



SOIL BIOLOGY 



PRACTICE 1. 

EXAMINATION OF MICROORGANISMS IN 
SOILS AND MANURES. 

Many kinds of microorganisms inhabit soils and ma- 
nures. Their presence in soils is essential to agriculture 
in general since the soil is the basis of all agriculture. It 
is also true that life would cease on the earth were it not 
for the activity of these organisms. 

Study the typical forms as they occur in their natural 
media as outlined below : 

(a) Prepare an infusion of each of the following samples: 

1. Fresh horse manure. 

2. Sandy loam. 

3. Rich loam. 

Place 50 grams in 100 cc. of sterile water contained in 
a 200 cc. sterile Erlenmeyer flask. Shake vigorously for 
5 minutes and then allow it to stand until after the follow- 
ing procedure has been carefully carried out. 

(6) 1. Place the microscope on the table in a position which per- 
mits of comfortable use. 

2. Bring the draw tube to standard length. 

3. Remove the eyepiece and arrange the plane mirror, using 

Abbe condenser, so that the field of light is clear and free 
from obstructions such as window bars, trees, etc. 
- 4. The illumination should be central. (Transmitted axial 
light.) 
5. Examine with medium power a specimen of algae, diatom, 
a sand or soil grain, cotton fiber or an air-bubble. 
3 



4 SOIL BIOLOGY 

6. Change the illumination to oblique (transmitted oblique 

light) by placing the finger below and half over the light 
opening of the iris diaphragm of the condenser. Note 
the advantage of oblique light for surface and mor- 
phological studies. 

7. Repeat numbers 5 and 6, varying the opening of the iris 

diaphragm as follows: Wide open, open |, and f the 
diameter of the rear lens opening of the objective. 
This is determined by removing eyepiece and looking 
into the tube. 

8. Focus oil immersion objective and lower it with coarse 

adjustment until it comes into contact with the oil. 
Determine this by watching carefully with the head 
to one side, complete focusing with fine adjustment. 
Always focus upward when looking into the instrument. 

Examine each in the following manner: By means of a 
sterile glass rod remove two drops of the liquid. Place it 
upon a clean slide. Examine with low, medium, and oil 
immersion objectives, using a cover slip. In a similar 
manner, make stained preparations on the slides, using 
carbol-fuchsin, methylene blue, gentian violet, and iodine, 
and examine with oil immersion objective. Do not use 
cover slips for the stains on the slides. INote the different 
forms present and which one predominates. Examine for 
cells of higher plants, fungous growths (mycelial and 
sporal), algae, diatoms, and protozoa (amoebae, ciliates, 
flagellates). 



MICROORGANISMS 



Material 


Different forms 
found 


Predominant class 
of bacteria 




Bacillus 


Coccus 


Spirillum 













References. 

1. U. S. Dept. Agr., O. E. S., Bui. 194, 6, 7, and 13. 

2. Read the booklet accompanying your microscope. 

3. Elementary Chemical Microscopy, Chamot (1915), 1-53, 
102-158. 

4. Bacteriological methods, pages 91-93, this manual. 



Questions. 

-1. Which of the microorganisms found are vegetable and which 
animal? 

2. Discuss the distribution of bacteria in soils. 

3. How do the microorganisms obtain their food in a soU? 

4. Name the sources of the twelve essential elements for soil 
microorganisms. 

5. Which class of the microorganisms is the most important 
and why? 



PRACTICE 2. 

OCCURRENCE OF BACTERIA AT DIFFERENT 
DEPTHS IN SOILS. 

QUANTITATIVE BACTERIOLOGICAL EXAMINATION OF 
SOILS. 

Fresh samples of the surface soil (6f inches) and the 
subsoil (at 35^0 inches) are collected in the manner pre- 
scribed on page 121. 

Place 100 grams of the soil in a sterile 400 cc. shaker 
bottle and add 200 cc. of sterile water. Submit the mix- 
ture to five minutes shaking in the mechanical shaker or 
by hand. This soil infusion is used for inoculating pur- 
poses. Allow it to settle 15 minutes to facilitate measur- 
ing. By means of sterile pipettes make the following 
dilutions : 

2 cc. of the infusion in 98 cc. of sterile water (A) 1-100. 
10 cc. of (A) into 90 cc. of sterile water (B) 1-1000. 
10 cc. of (B) into 90 cc. of sterile water (C) 1-10,000. 
10 cc. of (C) into 90 cc. of sterile water (D) 1-100,000. 
10 cc. of (D) into 90 cc. of sterUe water (E) 1-1,000,000. 
10 cc. of (E) into 90 cc. of sterile water (F)* 1-10,000,000. 
20 cc. of (F) into 80 cc. of sterile water ((?)* 1-50,000,000. 

Boiling flasks containing the correct amounts of sterile 
water will be found on the supply sheK. Letter the flasks 
as above. Place 1 cc. of dilutions (D), (E), (F), and (G), 
with sodium asparaginate or synthetic agar. Place the 
1 cc. in the sterile Petri dish and pour the agar quickly, 
tilting the dish to effect uniform seeding. Reserve dilu- 
tions (C) and (D) for further use in practices 3 and 4. 
When cool, place in the Petri dish containers and invert 

* (F) and ((?) not necessary if a poor soil. 
6 



BACTERIA 7 

the container. Place in room-temperature incubator. 
Count plates after 3-^ days. After counting, replace 
plates in incubator and allow the colonies to develop for 
a week or 10 days. Make further observations on the 
growth, using the Society of American Bacteriologists' 
Chart for descriptive notes. Consult laboratory charts 
for identifying colony characteristics. 

Each student should enter his results together with the 
results of another student using the same soil on the data 
sheet below. 



SOIL BIOLOGY 



Date 


Soil 
surface 


Me- 
dium 


Incubation 


Dilution 


Num- 
ber per 
1 CO. of 
dilution 


Av. per 

gram of 

soil 


No. 
per 
acre 


Notes 




Temp. 


Time 














































































































































































Average 













Sub-soil 




























































































































































































Average 











BACTERIA 9 

References. 

1. Agricultural Bacteriology, Percival (1910), 118-124. 

2. Iowa Exp. Sta., Research Bui. (1912), 8. 

3. U. S. Dept. Agr., O. E. S., Bid. 194, 8-13. 

Problems. 

1. Calculate in pounds per acre the dry weight of the bacteria 
found in this soil. 

500 miUion dry bacteria weigh 0.2 milligram, living 1 milligram. 

2. Calculate in cubic feet the volume occupied by the living 
bacteria in this soil. 

100 million occupy 0.2 cubic millimeter. 

3. How many pounds of nitrogen and phosphorus are contained 
in the bacterial bodies of ah acre, as based upon the dry weight 
figures obtained mider number one ? 

Analysis of Bacteria. 

(Dry Basis.) 
Nitrogen 2.3 per cent. Phosphorus 1.2 per cent. 

Questions. 

1. Do numbers of bacteria in a soil indicate efficiency as to 
biochemical reactions? 

2. Explain the differences fovind between the surface and subsoil? 

3. How does the number of bacteria compare with the number of 
soil particles in a gram of a silt loam? Explain the reason for this 
difference. 



PRACTICE 3. 

OCCURRENCE OF NON-VEGETATIVE FORMS OF 
BACTERIA AND FUNGI IN SOILS. 

The number of non-vegetative forms is determined in 
samples of the surface soil of the same type as that used in 
the previous practices. Add to each tube of melted agar 
(synthetic or sodium asparaginate) and melted fungi 
gelatin 1 cc. of dilution (d). Heat duplicate tubes of each 
medium at the following temperatures, 70°, 85°, and 
100° C. for ten minutes. Pour plates at 40-42° C. and, 
when cool, place in Petri dish container, invert and place 
them in the room-temperature incubator. Examine at the 
end of two days. Count in the manner already described 
after 3-4 days. Return the plates to the incubator and 
allow the colonies to further develop for 2 weeks or longer. 
Note pigment formation and colony characteristics. 
Observe which form disappears at the various tempera- 
tures. Calculate the percentage of the total number that 
are in the non vegetative stage. 



10 



BACTERIA And fungi 



11 



Date 


Soil 


Me- 
dium 


Incubation 


Dilu- 
tion 


Heated 

to 

temp. 


Num- 
ber per 
1 cc. of 
dilu- 
tion 


Av. 

per 
gram 
of soil 


No. 
per 
acre 


Notes 


Temp. 


Time 











































































































































































































































































Question. 

1. How do you explain the presence of the non-vegetative forms 
in normal soils? 



PRACTICE 4. 

OCCURRENCE OF FUNGI AT DIFFERENT DEPTHS 
AND IN DIFFERENT SOILS. 

Prepare 1-10,000 dilutions of the surface soils and 
1-1000 dilutions of the subsoils used in practice 2. Plate 
in duplicate 1 cc. of each dilution employing the fungi 
gelatin. Incubate at room temperature for 3-4 days or 
until the colonies are easily recognized as fungi. Make 
the counts at this time, using the binocular or hand lens. 
Study the microscopic appearance of the colony. Draw 
the typical colonies under the binocular. Allow the 
fruiting bodies to mature. Examine the mycelia and 
fruiting bodies carefully. Which are septate or non- 
septate? Are the fruiting bodies perfect or imperfect? 
The I objective is used for the fruiting bodies. 



Date 


Soil 


Mer 
dium 


Incubation 


Dilu- 
tion 


Depth 


No. 
per 
cc. 


Av. 

per 

gram 


No. 
per 
acre 


Notes 


Temp. 


Time 







































































































































References. 

1. Household Bacteriology, Buchanan (1913), 50-84, 487-523. 

2. Cornell Agr, Expt. Sta., Bui. 315 (1912), 415-419, 437-501. 

12 



OCCURRENCE OF FUNGI 13 

Questions. 

1. What kinds of fungi inhabit soils? 

2. Which of these kinds predominate? 

3. Of the factors necessary for growth which are most important 
for soil fungi? 

4. In the struggle for food how are soil fungi at an advantage? 



PRACTICE 5. 

AMMONIFICATION IN SOILS.* 

INFLUENCE OF MOISTURE CONTENT ON THE PROCESS. 

Ammonification, which is the production of ammonia 
from organic compounds by microorganisms, is greatly 
influenced by the moisture content of a soil. There is an 
optimum moisture content for all bacterial activities in 
soils and it is determined as outlined in this exercise with 
the exception that periodic determinations are usually 
made while here one suffices to show the influence the 
moisture factor exerts on this process. 

On the bulletin board will be posted the soil type, the 
organic matter (kind and amount) and the amount of 
sterile water to add to each treatment in addition to that 
indicated below. 

Weigh ten 100 gram portions of the air-dry sieved soil 
into the jelly glasses. Add the organic matter with a 
sterile spatula and thoroughly mix. Add the sterile 
water as indicated below with a sterile pipette slowly and 
evenly throughout the entire mass. Leave the surface 
level. 

1 and 2 6 cc. 

3 and 4 12 cc. 

5 and 6 18 cc. 

7 and 8 24 cc. 

9 and 10 30 cc. 

* Organisms concerned in the liberation of ammonia from or- 
ganic compounds are not isolated in this course as they have been 
studied in the general course in bacteriology. They are furnished 
to those students who wish to further study them from pure cultures. 

14 



AMMONIFICATION IN SOILS 



15 



Tlace the tin covers on the glasses and set them in the 
room-temperature incubator. After 14 days remove and 
transfer contents to a Kjeldahl flask. Determine am- 
monia by direct distillation with magnesium oxide as 
outlined on page 112. 



Group 

Blank on Method mgs. 



Soil Type 

1 cc. NH4OH mgs. N. 



Date 


Sample 
num- 
ber 


Period 
of 

incuba- 
tion, 
days 


Treat- 
ment 


HCl < 


= NH40H 


Titrated 

back, 
NH4OH 


Equiv- 
alent 
in sam- 
ple, 
NH4OH 


Mgs. 

N 


Notes 














































































































































- 





























































References. 

1. Consult the data sheets on the reference shelf. 



Pfoblems. 

1. Prepare a graph on cross-section paper, the abscissas repre- 
senting percentages of moisture, the ordinates percentages of nitro- 
gen recovered of the initial applied. 

Nitrogen content of the materials used wiU be foimd on the 
bulletin board. 



16 SOIL BIOLOGY 

2. Show by chemical equations the reactions which yield am- 
monia from proteia, protein derivatives, amides, and amino acids. 

3. ' Tabulate in your laboratory manual the names of ten typical 
ammomfiers. 

Questions. 

1. Ammonification is the result of cell activity; what are the 
active agents which enable the cell to assimilate and digest organic 
material? 

2. How does moisture influence these agents? 

3. What farm crops are able to utilize ammonia directly? 

4. How does ground limestone and rock phosphate influence 
ammonia production? 

5. What is the normal ammonia content of the corn-belt soil? 

6. What influence does the mechanical composition of a soil exert 
on ammonification? 

7. How does chemical composition influence ammonification? 

8. What effects do the biological factors have on ammonification? 



PRACTICE 6. 

AMMONIFICATION OF UREA AND ISOLATION 
OF UREA ORGANISMS. 

Place 20 cc. of urea solution in each of six 200 cc. Erlen- 
meyer flasks, plug and sterilize in the autoclave at 10 
pounds pressure for 10 minutes. 

Inoculate as follows : 

1 and 2, nothing (sterile). 

3 and 4, 1 gram of fresh soil. 

5 and 6, 1 gram of fresh horse-manure. 

Place in the incubator at room temperature and after 
48 hours remove 5 cc. of each treatment with a sterile 
pipette, filter and titrate against standard acids. Use 
weak acid for No. 1 and 2, strong acid for the others. 
Make a second titration in a similar manner 24 hours 
later. Calculate the per cent of urea changed at each 
period. 

Isolation of Urea Organisms. ■ — Pour plates of each 
of the soil and manure treatments by transferring a loopf ul 
to 10 cc. of sterile water. From these dilutions, inoculate 
tubes of sterile, liquefied, and cooled (40° C.) urea agar. 
Plate as usual. Incubate 2-4 days at 20° C. and transfer 
after 4 days to other plates and further transfer until pure 
cultures are obtained. 

Stain the organisms and examine in a hanging drop. 
Describe them carefully as to size, shape, motility, and 
rateof ammonia production. Inoculate tubes containing 
10 cc. of sterile urea solution with a loopful from typical 
colonies. Note turbidity. Titrate to obtain ammonia 
production at end of 2 days. Determine whether the 

17 



18 



SOIL BIOLOGY 



animonia is produced under aerobic or anaerobic conditions 
by heating 10 cc. of urea solution to expel dissolved gases; 
cool and inoculate, immediately covering the surface with 
1 inch of heavy oil and plugging the test tube tightly with 
cotton. 

Group Soil Type 

Blank on Method mgs. N. 1 cc. NH4OH mgs. N 



Date 


Sample 
num- 
ber 


Period 
of incu- 
bation, 
days 


Treat- 
ment 


HCl s 


= NH40H 


Titrated 

back, 
NH4OH 


Equiva- 
lent in 
sample, 
NH4OH 


"ff- 


Notes 











































































































































































































85. 



References. 

1. Handbuch der technischen Mykologie, Lafar (1904-6), 3, 71- 

2. Centbl. f. Bakt. 2 Abt. (1913-14), 39, 209-358, plates after 358. 



Problem. 

1. Calculate how many pounds of nitrogen, as nitrate, are possible 
from the lu-ea occurring in the urine of one cow, for a year, if 20 
pounds of urine are voided per day. 



UREA 19 

Questions. 

1. Write the chemical reactions showing the ammomfication of 
urea and the calories yielded. 

2. Are the common ammonifiers able to decompose urea? 

3. Of what special imporiiance are the urea organisms? 



PRACTICE 7. 

NITRITATION. 

OXIDATION OF AMMONIA TO NITRITE BY NITROSOMONAS. 

Influence Exerted by Carbonates, Ignited Soil, 
Soluble Organic Matter, and Aeration. 

(This experiment also illustrates denitrification.) 

Place 25 cc. of the salt solution for nitrite formation 
(page 95) in each of 12 one-liter Erlenmeyer flasks (ratio 
of depth to diameter 1 : 20-22). 

Make the following additions: 

1 and 2, nothing. 

3 and 4, 1 gram ground limestone or dolomite. 

5 and 6, 1 gram magnesium carbonate. 

7 and 8, 50 grams ignited soil. 

9 and 10, 50 grams ignited soil + 1 gram limestone or 

dolomite. 
11 and 12, 0.5 gram dextrose + 1 gram limestone or 

dolomite. 

Some students will perform this practice using 100 cc. 
flasks (ratio, depth to diameter, 1 : 3-4), plugging tightly 
with cotton. Plug the flasks loosely and sterilize in the 
autoclave at 12 pounds pressure for 15 minutes. When 
cool, add with a sterile graduated pipette the required 
amount of a standard solution of ammonium sulfate, 
carbonate, or nitrate, to give 10 milligrams of nitrogen per 
flask. 

Inoculate each with 1 gram of fresh soil obtained pref- 
erably from some continuous soil or crop experiment. 
Place flasks in the 30° or the room-temperature incubator 

20 



NITRITATION 



21 



as indicated by instructor. At the end of 2, 3, and 4 weeks 
test all for nitrite by the method outlined on page 116. 
Show each test to the instructor. The quantitative 
determination will be made upon advice of the instructor. 
Determine the nitrite in 1-10 inclusive by the per- 
manganate method (see page 113). 

Group Soil Type 

Blank on Method. . .mgs. N. 1 cc. N/10 KMn04 mgs. N. 



No. 


Treat- 
ment 


cc. 

N/10 
KMn04 
taken 


cc. 

N/10 
NaS203 
titrated 


Equiv- 
alent 
N/10 

KMn04 


Mgs. 

N 


Per cent 
of initial 
nitrogen 
changed 


Qualitative 

tests 




- 


- 


- 


- 


- 


- 


- 




















































































































„ 














- 


- 


- 


- 


- 


- 


- 





































References. 

1. U. S. Dept. Agr., O. E. S., Bui. 194, 57-67. 

2. Agricultural Bacteriology, Percival (1910), 134-168. 

3. N. J. Agr. Exp. Sta. Ann. Kept. (1908), 29, 117-119. 

Questions. 

1. Write the reaction for this oxidation, vising ammonium sul- 
fate, carbonate, and nitrate in the presence of calcium carbonate. 

2. Do nitrite exist in normal soUs? 

3. Are nitrites assimilable by plants? 



22 SOIL BIOLOGY 

4. What three factors are most detrimental to the growth of this 
organism mider field conditions? 

5. Give the notable exception exhibited by this organism in its 
nutrition. 

6. What is the chief som-ce of this element? 

7. Name 10 elements which wiU suflSce to neutralize the acid 
formed. 

8. What substances retard and check nitritation and in what 
concentrations? 

9. What substances accelerate nitritation? 

10. Discuss the importance of nitrite production from the stand- 
point of the liberation of insoluble minerals. 



PRACTICE 8. 

NITRATATION. 

OXIDATION OF NITRITE TO NITRATE BY NITROBACTER. 

Influence Exerted by Carbonates, Ignited Soil, 
Soluble Organic Matter, and Aeration. 

{This experiment further illustrates denitrification.) 

Place 25 cc. of the salt solution for nitrate formation 
(page 95) in each of 12 one-liter flasks. Make the 
following additions: 

1 and 2, nothing. 

3 and 4, 1 gram ground limestone or dolomite. 
5 and 6, 1 gram magnesium carbonate. 
7 and 8, 0.025 gram sodium carbonate. 
9 and 10, 50 grams ignited soil. 
11 and 12, 0.5 gram dextrose + 1 gram ground lime- 
stone or dolomite. 

As in the previous practice, this practice will also be 
conducted with 100 cc. flasks plugged tightly with cotton. 
Plug flasks loosely and sterilize in the autoclave at 12 
pounds pressure for 15 minutes. When cool add to each 
the required amount of standard sodium nitrite solution 
which gives 10 milligrams of nitrogen per flask. 

Inoculate each flask with 1 gram of soil as in the previous 
practice. Place flasks in the 30° or room-temperature 
incubator as indicated by instructor. At the end of one 
week test quahtatively for nitrate by the method given 
on page 117. At the end of 2 weeks test for nitrites by 
the method used in the previous practice and then deter- 
mine nitrate nitrogen in all but 11 and 12 by the method 
given on page 113. 

23 



24 



SOIL BIOLOGY 



Group Soil Type . . . . 

Blank on Method Mes. 1 cc. NH4OH. 



.Mgs. 



No. 


Treat- 
ment 


HCI < 


NH4OH 


Titrated 

back, 
NH4OH 


Equiva- 
lent in 
sample, 

NH4OH 


Mgs. 

N 


Per cent 
of ini- 
tial N 

changed 


Qualita- 
tive tests 




- 


- 


- 


- 

























































































































































































References. 



1. Centbl. f. Bakt. 2 Abt. (1899), 5, 329, 377, 429. 

2. Centbl. f. Bakt. 2 Abt. (1910), 27, 169. 

3. Science (1912), 35, 996. 

4. Expt. Sta. Record (1908-9), 20, 518, 519-520. 

5. Ohio Expt. Sta. Bui. Tech. Series (1915), 7. 

6. Ann. Inst. Pasteur (1904), 18, 181-196. 

Problem. 

1. Calculate the pounds of carbon dioxid reduced when 35 pounds 
of nitrogen are oxidized. 

Questions 

1. What is the function of the base in this reaction? 

2. Explain the effect of sodium carbonate. 

3. How is the effect represented by the action of sodium carbon- 
ate obviated under field conditions? 



NITRATATION 25 

4. What is the cause of the action produced by the ignited soil? 

5. Name ten nitrites which are changed to nitrates. 

6. What substances retard or check nitratation, and in what con- 
centrations ? 

7. What substances accelerate nitratation? 

8. Write the reaction for this transformation. 

9. Discuss the value of nitrates compared with nitrites, ammonia, 
and soluble organic nitrogen in soils. 



PRACTICE 9. 

ISOLATION AND STUDY IN PURE CULTURE OF 
NITROSOMONAS AND NITROBACTER. 

In the isolation of the nitrite and nitrate organisms 
advantage is taken of the already vigorous growths exist- 
ing in treatments 9 and 10 of practices 7 and 8. When 
the qualitative tests have given a strong reaction (consult 
instructor for advice at this time), follow the procedure 
outhne below. 

Isolation of NiTROSOMONAS. — Procedure: 

1. Place 1 cc. of the solution (numbers 7 and 8 in prac- 
tice 7) in a sterile Petri dish. 

2. Add 10 cc. of sterile silica jelly (Solution I) in the 
Petri dish and thoroughly mix. 

3. Add 1 cc. of sterile solution of sodium carbonate and 
ammonium sulfate (Solution II), and immediately tilt to 
mix the contents of the dish as the jelly solidifies rapidly. 

Caution should be exercised in the manipulation de- 
scribed under 3 as rough plates resulting from uneven 
mixing of the carbonate solution with the jelly make it 
■difficult to see the colonies. The plates should be left 
level until solid. An excess of moisture is undesirable 
and will not occur if the above procedure is followed. 
After the medium has become solid, label the plates and 
place them in Petri dish containers and incubate at 30° C. 
AHow the colonies to develop until the centers become 
yellow or orange, when the microscopical study should 
begin. Test colonies with nitrite and nitrate reagents 
before transferring. Transfer from typical colonies to 
magnesium ammonium phosphate agar. When typical 
colonies are well developed (4-6 days), transfer to the 

26 



NITROSOMONAS ' 27 

sterile magnesium plaster of Paris blocks, which are half 
submerged in a solution for nitrite formation, in Petri 
dishes. Inoculate sihca jelly and agar slants. Study the 
colony characteristic on the various media. Stain the or- 
ganism with carbol-fuschin, gentian violet, and methylene 
blue. Study the size and shape, and compare with nitrate 
organism. Prepare a permanent slide. Inoculate a small 
sterile flask containing ignited soil and solution for nitrite 
formation and incubate at 30° C. Test for nitrite pro- 
duction at the end of 5 days. This method together with 
the microscopic study will determine if your culture is 
pure. 

Isolation of Nitrobacter. — Proceed as in the isola- 
tion of Nitrosomonas, using instead inoculating solution 
from 9 or 10, practice 8, and 1 cc. of a solution of sodium 
carbonate and sodium nitrite (Solution III). Omit the 
use of magnesium ammonium phosphate agar, otherwise 
make similar studies with this organism and inoculate a 
small flask after studying the organism in pure culture. 

References. 

1. Exp. Sta. Rec. (1890), 2, 751-757 (Winogradsky). 
- 2. Jour. Chem. Soc. (London) (1878), 33, 44 (1898), 59, 484 
(Warington) . 

3. Jour. Chem. Soc. (London) (1891), 60, 352 (Franklands). 

Questions. 

1. From the qualitative tests which organism is the more rapid 
grower and what are the comparative rates of oxidation? 

2. Give the group number for both these organisms (Soc. Am. 
Bact. Chart). 



PRACTICE 10. 

PRODUCTION OF INORGANIC NITROGEN IN 

SOILS. 

COMPARATIVE AMMONIFICATION AND NITRATATION OF 

CROP RESroUES, GREEN MANURES, AND FARM 

MANURES IN TYPICAL SOILS. 

Influence Exerted by Carbonates, Soil Type, Mois- 
ture, AND THE Condition of the Organic Sub- 
stances. 

This experiment is designed to show the weekly am- 
monia and nitrate production from organic materials, 
such as are used in agricultural practice and under as 
nearty similar conditions as possible. Such materials as 
clover, sweet clover, soybeans, cowpea, and alfalfa hays, 
corn stalks, wheat and oat straw, and farm manures are 
applied in both the green and dry condition according to 
common usage. 

It would be advisable to use fresh soil for this practice 
if convenient. The materials applied in the dry condition 
should all pass a 10-mesh sieve. Green materials may be 
applied much coarser. 

This practice is conducted in groups. Students are 
permitted to use soils from their own farms or ones in 
which they are particularly interested. Each group of 
students conducts four sets of treatment on a given soil. 

Weigh out fifty-six 100 gram portions of the soil to be 
studied. Place in the jelly glasses and make the following 
applications : 
1-14, nothing. 

15-28, 1 gram of carbonate (limestone or dolomite). 

29-42, organic matter. 

43-56, 1 gram carbonate + organic matter. 

28 



INORGANIC ' NITROGEN 29 

The students are permitted to choose the kind of organic 
matter they desire to test and the amomits to be added 
are posted. 

The sterile water to be added will be found on the 
bulletin board and is the optimum for the various treat- 
ments. 

Mix thoroughly and place glasses in the room-tempera- 
ture incubator. Each week 1-3 cc. of sterile water 'is 
added to each glass to compensate for loss due to evapora- 
tion. 

Ammonia and nitrate determinations are made on du- 
plicates of all treatments at the beginning of the experiment, 
and every 7 days, for 7-10 weeks.* 

The sample is divided into two equal parts by weight. 
On one-half determine the ammonia by direct distillation 
or by aeration. Dry the other half at 108° C. for 6-8 
hours in the electric oven, then add 300 cc. of dilute hydro- 
chloric acid (5 cc. per liter). Shake vigorously several 
times. Allow the solution to settle a few minutes when 
200 cc. is removed by suction. Proceed as indicated for 
the determination of nitrites and nitrates, page 113. 

If the total nitrogen content is not known it will be 
-necessary to determine it on the soil used. The total 
nitrogen content of the typical soil types and the organic 
materials will be posted. 

* It is sometimes convenient to make the intervals 9 and 1 1 days 
to conform to the laboratory periods. 

Other materials such as raw rock phosphate are used in this 
experiment. The number of jelly glasses may be increased to 
extend through the growing period of a crop, 



30 



SOIL BIOLOGY 



AMMONIA DETERMINATIONS. 



Date 


Sample 
No. 


Treat- 
ment 


HCl ^ 


SNH4OH 


Titrated 

back, 
NH4OH 


Equiva- 
lent in 
sample, 
NH4OH 


Mgs. 


P.P.M. 


Notes 



















































































































































































































































































































































































































































































































































































INORGANIC NITROGEN 



31 



AMMONIA DETERMINATIONS. 



Date 


Sample 
No. 


Treat- 
ment 


HCl =: 


= NH40H 


Titrated 

back, 
NH4OH 


Equiva- 
lent in 
sample, 
NH4OH 


Mgs. 
N 


P.P.M. 


Notes 






















































































































































































































































































































































- 





























































































































































































































32 



SOIL BIOLOGY 



NITRATE DETERMINATIONS. 



1 

p 


fl 

02 a 


1 

S 
£ 


1 

1 




5 , 








IS 

1 


PM 


1 
1 



































































































































































































































































































































































































































































































































































































































































































INORGANIC NITROGEN 



33 



NITRATE DETERMINATIONS, 



p 


sa 


a 

<D 

s 

1 


'3 

h 

1 




_o 

o 
o 










2; 

1^ 




1 
1 


















































































































































































































































































































































> 
















































^ 








































































































































































































































































■ 

























34 



SOIL BIOLOGY 



SUMMARY OF THE NITROGEN DETERMINATIONS. 



Group . 
Group . 



Soil Type . 
Soil Type . 



Treat- 
ment 


Nitrogen 

as 


Parts per million of nitrogen in 
water-free soil 


Pounds 


Date of determinations 


per 
acre 




Begin- 
ning 


















Av. 






Nitrate 
Ammonia 


























Nitrate 
Ammonia 


























Nitrate 
Ammonia 


























Nitrate 
Ammonia 


























Nitrate 
Ammonia 


























Nitrate 
Ammonia 


























Nitrate 
Ammonia 


























Nitrate 
Ammonia 

























References. 

1. Soil Fertility and Permanent Agriculture, Hopkins, 194-198. 

2. Soil Conditions and Plant Growth, Russell (1913), 78-89. 

3. Centbl. f. Bakt. 2 Abt. (1908), 25, 64. 

4. Centbl. f. Bakt. 2 Abt. (1910), 27, 169-186. Exp. Sta. Reed. 

(1910), 23, 721. 

5. Hawaii Agr. Exp. Sta. Bui. 39 (1915), 24-25. 

6. Jour. Indus. & Eng. Chem. (1915), 7, 521. 

7. N. J. Rept. of Soil Chemist and Bacteriologist (1914), 217, 220. 

Problems. 

1. Plot the ammonia and nitrate data for all treatments on cross- 
section paper, the abscissas representing the time and the ordinates 
the milligrams of nitrogen as ammonia and nitrate. Use red for 
nitrate, blue for ammonia and solid, broken, single, and double 
dotted lines for the treatments. 



INORGANIC NITROGEN 35 

2. Explain the fluctuations of both the ammonia and nitrate 
curves. 

3. Calculate the yield of oats, wheat, and corn possible from the 
nitrate and ammonia found. 

4. Correlate the ammonia and nitrate production on the soil used 
with crop yields. 

Questions. 

1. Under field conditions is there need of an application of 
nitrate nitrogen? 

2. Does nitratation go on in acid soils? 

3. Do non-nitrifiable soils exist? 

4. How should a non-nitrifiable soil be treated? 

5. How does the crop influence nitrate and ammonia formation? 

6. What effect does cultivation have on nitratation? 

7. Are nitrifying organisms active in the fall and winter months? 

8. What means may be used to check the loss of ammonia and 
nitrate from soils? 

9. Is the loss of ammonia from the brown silt loam appreciable? 

10. Why does organic matter not inhibit the growth and ac- 
tivity of these organisms in soils? 

11. What factors are most important in nitrate production in 
field soils? 

12. What factors are most important in ammonia production in 
field soils? 

13. What is the average annual loss of nitrogen per acre? 
_ 14. Is there any nitrification below the surface soil? 

15. How does the nitrate content of a soil vary from the surface 
soil to a depth of 5 feet? 



PRACTICE 11. 
CARBON DIOXID PRODUCTION. 

Carbon dioxid is produced by respiration of the micro- 
organisms in soils. It is evolved from soil into the air in 
large amounts. A large amount of carbon dioxid bathes 
the soil and liberates insoluble elements by the production 
of acid and salts. The importance of the carbon cycle is 
understood. 

The rate and amount of carbon dioxid evolved depends 
upon many factors, chiefly the kind, amount, and stage of 
decomposition of organic matter present in the soil. A 
determination of nitrogen as ammonia and nitrate makes 
possible a calculation of the carbon nitrogen ratio of de- 
composition. 

Place six 100-gram portions of the soil to be tested in a 
beaker and add the organic materials. Thoroughly mix 
and place in 500 cc. Erlenmeyer flasks equipped with glass 
tubes on the end of which are rubber tubings which may 
be opened for the admission of air and a pair of Wort- 
mann valves (Greiner and Friedricks, Cat. No. 3459), one 
above the other. Into both valves place 10 cc. of stand- 
ard potassium hydroxid. The carbon dioxid from the 
soil is collected in the lower valve, while the upper valve 
serves as a trap collecting carbon dioxid from the air. 

Arrange as follows : 

1 and 2, soil alone. 

3 and 4, soil + 2 grams dextrose. 

5 and 6, soil + 2 grams organic matter. 

Place the stopper containing the glass tube and valves in 
the flask and allow the flasks to remain at room tempera- 
ture in a reasonably shaded place (dark not necessary), 

36 



CARBON DIOXID .PRODUCTION 



37 



Every two days remove the valves and titrate the 
contents of the lower one as indicated on page 12. At the 
end determine the ammonia on 50 grams and the nitrate 
on 50 grams of treatment numbers 5 and 6. 

Group Soil Type 

lOcc. KOH cc. . .Acid 1 cc. KOH mgs. CO2 



Num- 
ber 


Treat- 
ment 


Milligrams carbon 
dioxid 


P.P.M. 

total 
CO2 


P.P.M. 
NH3 


P.P.M. 
NO3 


CO2/NO3 
ratio 


Days 




- 


- 


- 





- 


~ 




- 


- 


- 
































- 


- 


- 






















f-' 
















- 


- 


- 






















' 





































References. 

1. Soil Fertility and Permanent Agriculture, Hopkins, 33. 

2. Soil Conditions and Plant Growth, Russell (1913), 78. 

3. lU. Agr. Exp. Sta. Bui. 145, 105-111, 121. 

4. U. S. Dept. Agr., O. E. S. Bui. 194, 55-56. 

5. Iowa Exp. Sta. Research Bui. 3, 135-154. 

6. Jour. Agr.'Sci. (1915), 7, 44. 

7. Centbl. f. Bakt. 2 Abt. (1910), 28, 45. 



38 SOIL BIOLOGY 

Problems. 

1. Calculate how long the carbon supply of the atmosphere over 
an acre would be sufficient for 100 bushel, crops of corn if micro- 
organisms failed to maintain the supply. 

2. From the figures obtained in this practice, what can be 
deduced as to the stage of decomposition of the soil and the organic 
matter used? Indicate this by carbon, nitrogen ratios. 

Questions. 

1. How does carbon dioxid originate from soil organic matter? 

2. WMch classes of organisms are most active in carbon dioxid 
production and under what conditions? 

3. What per cent of carbon dioxid is found in a normal soil , 
atmosphere? 

4. What factors are important in the fluctuations occurring in 
the carbon dioxid content of the soil atmosphere? 

5. In what way does carbon dioxid prove injurious? 

6. What beneficial action does it produce? 

7. How would you overcome the injury arising from planting 
immediately after plowing under a green crop? 



PRACTICE 12. 

CELLULOSE DECOMPOSITION. 

AEROBIC DECOMPOSITION BY FUNGI AND BACTERIA. 

Contrary to earlier conceptions, the decomposition of 
cellulose and fibroiis residues takes place very actively 
under aerobic conditions. Fungi play a very important 
role in this decomposition. 

Place 2 grams of filter paper, cut into squares of about 
I of an inch, and 2 grams of corn stover or straw in jelly 
glasses with 100 grams of soil. Add sterile water to make 
the optimum moisture content. Place all in the 30° C. 
incubator for 10-14 days, observing the fungous growth 
at frequent intervals. 

1 and 2, paper 2 grams. 

3 and 4, straw or stover 2 grams. 

5 and 6, soil, only. 

Plate from 1, 3, and 5 on cellulose and starch agar. 
Incubate at 30° C. for 3 weeks, then transfer to cellulose 
and starch agar. At this time expose plates of cellulose 
and agar to the laboratory air for 5 minutes. Study the 
dissolving of the cellulose in the zone around the colony. 
Stain the organisms with carbol-fuchsin. Describe the 
organism and draw colonies and individuals. Allow 2, 
4, and 6 to remain 8-10 weeks, when an examination of 
the residual cellulose or stover should be made under the 
microscope. 

References. 

1. Centbl. f. Bakt. 2 Abt. (1904), 11, 689-695; (1908), 21, 700; 
(1909), 23, 300-304; (1910), 26, 222-227; (1910), 27, 1-7, 449-451, 
633. 

39 



40 SOIL BIOLOGY 

2. Centbl. f. Bakt. 2 Abt. (1912), 34, 63, 485-494. 

3. U. S. Dept. Agr. Bur. PL I. Bui. 266 (1913). 

4. SoU Science, 1916, 1, 437-487. 

Questions. 

1. What kinds of fungi decompose cellulose? 

2. What kinds of bacteria decompose cellulose? 

3. Is there a selective action exhibited by these organisms? 

4. What products are formed in the aerobic decomposition of 
cellulose? 

5. What is the enzyme concerned? 

6. Wni the cellulose of a green crop be more easily attacked than 
that of a dry drop? 

7. Name the important processes for which cellulose serves as a 
source of energy. 



PRACTICE 13. 
ANAEROBIC CELLULOSE DECOMPOSITION. 

In each of five 200 cc. Erlenmeyer flasks, place 150 cc. 
of solution for anaerobic cellulose decomposition. Weigh 
accurately 3 sets, of 3 each of 5| cc. diameter filter paper 
and place a set in each of three flasks, making sure that 
the papers are entirely submerged. In the other two 
flasks, place 3 grams of wheat or oat straw. Plug and 
sterilize at 12 pounds pressure for 10 minutes. 

Inoculate as follows: 

1, 2, and 3 with 5 cc. of a filtered infusion of well- 
rotted horse-manure. 
4 and 5 with 5 cc. of a filtered infusion from surface soil. 

Place in the room-temperature incubator. After de- 
composition has started, as judged by the frayed appear- 
ance of the edge of the paper, transfer, with a platinum 
needle, a small portion to a tube to be used in isolating the 
organism. 

Allow these flasks to remain several weeks or until such 
a time when the papers are apparently decomposed. 
Note the odor of hydrogen sulfide. Test with lead 
acetate paper. When decomposition has progressed suffi- 
ciently, filter the contents of the flasks on a weighed filter 
paper, wash with water, dry, and weigh. Report the loss 
of carbon. Isolate the organisms under anaerobic condi- 
tions, and test their ability to reduce sulfur compounds 
and to produce hydrogen sulfide from inorganic and 
organic sources. 

41 



42 



SOIL BIOLOGY 





Paper 


Straw 




1 


2 


3 


4 


5 




Initial weight 














Final weight , . . . 








Loss 


















Weight after drying 














Weight of filter paper used as 
filter 








Final weight 



















References. 

L Microbiology, Marshall (1912), 246-249. 

2. Vorlesungen tiber landwirtschaftliche Bakteriologie, Lohnis 
(1913), 171-177. 

3. Centbl. f. Bakt. 2 Abt. (1902), 8, 192-201, 225-231, 257-263, 
289-294, 321-326, 385-391 (Omelianski) ; (1904), 11, 369-377; 
(1904), 12, 33-43 (1906), 15, 673-687. 

4. Centbl. f. Bakt. 2 Abt. (1908), 20, 682; (1912), 34, 485-494. 

Problems. 

1. Calculate how many pounds of straw per acre will be de- 
composed under anaerobic conditions in 6 months at the rate found 
in this experiment. 



Questions. 

1. Which decomposes more rapidly the straw or pure cellulose? 

2. What are the chief products formed? 

3. What beneficial purposes may cellulose serve imder anaerobic 
conditions? 

4. Why is an excess of straw or coarse manure often very in- 
jurious to soil? 

5. For what 3 processes does cellulose serve as a source of energy 
under anaerobic conditions? 



PRACTICE 14. 

SYMBIOTIC NITROGEN FIXATION. 

INOCULATION OF LEGUME SEEDS. 

Growth of Nodules and Determination of 
Nitrogen Fixed. 

{B. radicicola is to he isolated from nodules obtained in this 
practice.) 

This practice may be omitted if the students have 
already had the work given in soil fertility or its equiva- 
lent. 

Place 12 pounds of washed sand in each of 6 one-gallon 
earthen jars and in two others place 10 pounds of brown 
silt loam. The jars may be rinsed with 1-300 mercuric 
chloride solution and then with sterile water if recently 
used for similar work. Select 5 of the larger legume seeds 
(cowpeas) and 15 of the smaller (clover) and steriUze by 
the method outlined on page 127. Arrange the jars as 
below, and place them in the greenhouse. 

1 and 2, inoculate with soil. 

3 and 4, inoculated by the glue method. 

5 and 6, uninoculated. 

7 and 8, inoculated (plant in soil). 

Plant the seeds in the usual manner and water with 
sterile water. Apply sterile plant food solution every ten 
days, omitting the nitrogen. After about 10-15 days, 
depending upon the legume, wash out the plants from 
numbers 1, 3, and 5, and examine the roots carefully. 
Study the nodules. Make a drawing showing the manner 
of attachment, shape, and size, and carefully describe the 

43 



44 



SOIL BIOLOGY 



appearance as to color and surface. Make cross and 
longitudinal sections and describe the internal appearance. 
Compare your description with that of other groups and 
report the differences found between the various legumes 
grown. After 20-30 days analyze 3 plants, either green 
or dry, of each of the inoculated and uninoculated, for 
total nitrogen. 

Remove small nodules from some of the plants washed 
out in 1 or 3 and proceed with practice 15. 



Sample 
No. 


Treat- 
ment 


HCl ^ 


-- NH4OH 


Titrated 

back, 
NH4OH 


Equiva- 
lent in 
sample, 
NH4OH 


Mgs. 


Gain 

from 

nodules 



































































































































References. 

1. 111. Agr. Exp. Sta. Cir. 86, Bui. 94. 

2. lU. Agr. Exp. Sta. Bui. 179, 471-482, 493-499, 503-522. 

3. Consult article dealing with Legume Inoculation (Reference 
Shelf). 

4. Vorlesungen iiber landwirtschaftliche Bakteriologie, Lohnia 
(1913), 361-378. 

Questions. 

1. Explain why the soil and glue methods are superior to the 
chemical cultures at the present time. 

2. Why is the glue method an ideal method? 



SYMBIOTIC NITROGEN FIXATION 45 

3. Explain the method of collecting, storing, and inoculating 
with soil. 

4. Name the legume groups as arranged for cross inoculating 
under field conditions. 

5. What percentage of the total nitrogen is derived from the air 
by inoculated legumes, on normal soils? 

6. Discuss the nitrogen enrichment of a sandy soU, a sUt soil, 
and a heavy clay soU by this symbiosis. 

7. Calculate the gain in nitrogen due to inoculation on the basis 
of standard crops of cowpeas and soy beans. 

8. Through what organs do legumes obtain the atmospheric 
nitrogen? 

9. What American scientists first noted and carefully studied 
nitrogen fixation? 



PRACTICE 15. 

ISOLATION OF B. RADICICOLA FROM LEGUME 
NODULES. 

If the previous practice was not conducted, proceed as 
follows: Plant the various legume seeds in both sand and 
soil in the one-gallon earthen jars, in the ordinary way, 
inoculating either by the glue method or by an infusion 
from the nodule. 

When the plants in the sand are well started, remove a 
small nodule with sterile forceps and place in a 1-500 mer- 
curic chloride solution for 3 minutes, remove and rinse in 
sterile water, pass into three test tubes of sterile water and 
finally crush in 1 cc. sterile water with a sterile glass rod 
or sterile forceps. Pour 5 loopfuls in a test tube of sac- 
charose-ash-agar, agitate thoroughly and remove 5 loop- 
fuls to a second tube of the agar. Pour these plates in 
the usual way and incubate at 30° C. It is essential to 
transfer several times on the saccharose-ash-agar before 
making the detailed study. After 10-12 days examine 
with a hand lens and describe the colony characteristics. 
Stain the organisms with aniline gentian violet and carbol- 
fuchsin. Examine in hanging drop. Describe the or- 
ganisms as to size, shape, motility, and reaction to stain. 
Make a permanent slide for your own collection. Ex- 
amine slides of other groups, and make notes on the 
organisms from the different legumes. 

Remove an old nodule from a legume plant growing in 
the soil and examine the organisms in the hanging drop. 

Draw the bacteroids as you see them. Stain the bac- 
teroids with carbol-f uchsin, heating to steaming twice with 
an interval of two minutes between. Examine some of 

46 



ISOLATION FROM B. RADICICOLA 47 

the bacteroids from the liquid-culture media which will be 
furnished. 

References. 

1. 111. Agr. Exp. Sta. Bui. 179, 482-488. 

2. Centbl. f. Bakt. 2 Abt. (1909), 23, 59-91. 

3. Vir. Agr. Exp. Sta. Ann. Rpt. (1909-10), 123-142, 145-174. 

4. Centbl. f. Bakt. 2 Abt. (1907), 19, 264-272, 426-441. 

Questions. 

1. Are these bacteroids an involution form? 

2. How do they differ from bacilli? 

3. In what form does the organism exist in the soU? 

4. How long are the organisms viable under field conditions? 

5. Does freezing of the soil kill the organisms? 

6. What effect does drying a soil have on these organisms? 

7. WUl these bacteria live in an acid soil? 

8. Are these organisms able to fix nitrogen without the legume 
plant? 

9. Discuss the importance of this symbiosis as related to a per- 
manent agriculture. 



PRACTICE 16. 
NON-SYMBIOTIC NITROGEN FIXATION. 

AEROBIC NITROGEN FIXATION IN SOILS. 

Influence of Carbonates on the Process. 

Fifty grams of soil (see posted sheet for soil type as- 
signed to your group) are placed in each of ten jelly 
glasses. Treat as follows: 

1 and 2, nothing. 

3 and 4, 0.3 gm. mannite. 

5 and 6, 0.3 gm. mannite and 1 gm. CaCOs. 

7 and 8, 15 gms. fresh clover or rye tops. 

9 and 10, rich algae slime, place in the direct light. 

The amount of water to be added will be found on the 
posted sheet. Place in the room-temperature incubator 
for four weeks, adding 3 cc. sterile water each week and 
stirring the soil in all biit 9 and 10 after adding it. At the 
end of this time dry the soil and grind it to pass 100-mesh 
sieve and determine the total nitrogen on 10-gram samples 
of each treatment. The total nitrogen content of the 
original soil before incubation together with the nitrogen 
content of the clover, algse, carbonate, etc., wiU be posted. 



48 



NON-SYMBIOTIC NITROGEN FIXATION 



49 



Group Soil Type 

Blank on Method mgs. N. 1 cc. NH4OH mgs. N. 



-2 
.Q 


■11 

il 

cog 


1 

1 




2; 






^ 

S 
S 




1 












































































































































































































References. 

1. Centbl. f. Bakt. 2 Abt. (1901), 7, 561-582. Centbl. f. Bakt. 
2 Abt. (1902), 9, 3-43. 

2. N. J. Exp. Sta. Ann. Rpt. (1903), 24, 217-286; (1904), 25, 
237-268; (1906), 27, 178-187; (1907), 28, 141-169; (1908), 29, 
137-147. 

3. Wis. Agr. Exp. Sta. Research Bui. 12 (1910). 

4. Col. Agr. Exp. Sta. Bui. 155. 

5. Soil Conditions and Plant Growth, Russell (1913), 93-95. 

6. lU. Agr. Exp. Sta. Bui". 179, 494. 



Problems. 

1. How many pounds of wheat straw, corn stover, and fresh 
clover tops must be oxidized to yield a fixation of nitrogen sufficient 
to supply a 50 bushel wheat crop and 100 bushel corn crop? 



50 SOIL BIOLOGY 

2. Calculate the fixation of nitrogen per acre, subtracting the 
results obtained in numbers 1 and 2 and the nitrogen content of 
the added materials from the various treatments. 

3. Explain the practical difficulties arising in determining the 
activity of Azotobader in normal soil, such as a brown silt loam. 

4. Explain the difference between Azotobader and B. radicicola 
in relation to organic carbon and the carbon cycle. 

Questions. 

1. Name the common Azotobader organisms. 

2. Why is isolation from a soil easier in the fall or winter? 

3. What influence does the reaction of the soil have on the growth 
of Azotobader? 

4. Name the chief sources of organic carbon for nitrogen fixation 
by Azotobader. 

5. What part may algae play in assisting Azotobader fix nitrogen? 

6. In what kind of soils does the greatest fixation occur? 

7. What organisms usually are found associated with Azoto- 
bader? 



PRACTICE 17. 

NITROGEN FIXATION IN SOLUTION BY SOIL 
BACTERIA. 

Place 25 cc. of solution for aerobic non-symbiotic nitro- 
gen fixation in each of ten • 450 cc. Erlenmeyer flasks. 
Add 20 grams of ignited soil, plug with cotton and sterilize 
at 12 pounds pressure for 15 minutes. Inoculate as below 
with one gram of soil. 

1 and 2, brown silt loam. , 
3 and 4, gray silt loam. 
5 and 6, brown sandy loam. 
7 and 8, yellow silt loam. 
9 and 10, nothing. 

Incubate at 30° C. for 25 days and do not disturb the 
scum any more than possible when transferring. Note 
any pecuHar odors during incubation. Analyze the con- 
tents of each flask for total nitrogen. Calculate the 
fixation. 



51 



52 



SOIL BIOLOGY 



Group 

Blank on Method . 



.mgs. N. 1 cc. NH4OH mgs. N. 





p 


E S 


a 
<u 

S 
1 




1^ 


15 W 




1 




1 


- 




























































































































































































. 













Reference. 

1. U. S. Dept. Agr., 0. E. S. Bui. 194, 8-15. 

Questions. 

1. What arguments have been advanced against this method of 
experimentation? 

2. What advantage has been claimed for this method? 

3. Give the general results of the workers who have shown this 
method to be less related to soil fertility than the methods which 
involve the soil instead of the solution. 



PRACTICE 18. 

ISOLATION OF AZOTOBACTER FROM SOIL. 

This practice is dependent upon practice 17. When a 
good scum has formed (7-10 days) transfer 3-5 loopfuls to 
a 100 cc. Erlenmeyer flask containing 20 grams ignited 
soil and sufficient solution for aerobic non-symbiotic 
nitrogen fixation, to half cover the soil slope. Likewise, 
transfer 2 or 3 times. After the second transfer pour 
plates of mannite agar, using 1 cc. of the solution for the 
first tube and 3 loopfuls from this for the second tube. 
Repeated transfers on the mannite agar are decidedly 
advantageous in studying the organism. Study the 
colony characteristics and stain the organism with carbol- 
fuchsin, gentian violet, methylene blue, and iodine. In 
using the iodine on the earlier preparations note any 
Clostridia forms. Make slants on this mannite agar. 
Study the multiplication of the organism and the zo- 
oglea. Measure the organisms. Classify the organism 
isolated. Describe carefully the pigment formation at 
the various stages. Grow the organism on the magnesium 
plaster of Paris block. Make permanent slides and draw- 
ings of the organism. 

References. 

1. Handbuch der technischen Mykologie, Lafar (1904-6), 3, 8, 
plates after 8. 

2. Vorlesungen iiber landwirtschaftliche Bakteriologie, Lohnis 
(1913), plates after 170. 

3. Centbl. f. Bakt. 2 Abt. (1913), 38, 14-25, plates after 24. 

4. Jour. Ag. Res. (1915), 4, 225-239, plates after 239. 
5: N. J. Agr. Exp. Sta. Ann. Rpt. (1908), 137. 

6. Jour. Agr. Sci. (1907), 2, 35-51. 

7. Jour. Agr. Res. (1916), 6, 675-702. 

53 



54 SOIL BIOLOGY 

Questions. 

1.- What advantage is derived from repeated transfers? 

2. Of what is the zooglea composed? 

3. What characteristic reaction does the brown pigment give and 
what erroneous conclusions might be drawn therefrom? 

4. How may the media on which growth occurs be responsible for 
differences in chemical analyses of bacteria? 

5. If combined nitrogen is present wiU it prevent the growth of 
Azotdbacter? 



PRACTICE 19. 

NON-SYMBIOTIC ANAEROBIC NITROGEN FIXA- 
TION AND ISOLATION OF B. CLOSTRIDIUM 
PASTEURIANUM. 

The fixation of nitrogen by this organism may in part 
counterbalance the activity of the denitrifying organisms 
which thrive under similar conditions. 

In each of 10 reduction test tubes, place 100 cc. of the 
solution for anaerobic nitrogen fixation. Plug and sterilize 
at 10 pounds pressure in the autoclave for 10 minutes. 
Obtain samples of soil at 6f , 20, and 40 inches and air dry 
in the dark. Heat the sample for 15 minutes at 75° C. 

1 and 2, 1 gram surface soil at 6f inches. 

3 and 4, 1 gram soil collected at 20 inches. 

5 and 6, 1 gram soil collected at 40 inches. 

7 and 8, surface soil saturated with the solution. 

9 and 10, solution sterile for transfers. 

' In 7 and 8 place enough surface soil to fill the 1 inch 
of the top, add solution to completely saturate the soil. 
Tubes 9 and 10 are to be used for transfers. 

Pour oil on the surface of 1-8 inclusive (about ^ inch of 
paraflin oil), plug tightly, and incubate at 30° C. Trans- 
fer from 3 and 4 to 9 after examining to see how growth 
has developed. Make transfers until cultures are pure. 
Study the organism by the usual methods. Stain with 
iodine solution. Note -carefully a characteristic odor. 
Determine total nitrogen on 1-6 and consult bulletin 
board for nitrogen control. Calculate the fixation. 



55 



56 

Group . 



SOIL BIOLOGY 

1 cc. NH4OH .mgs. N. 



1 

Q 


c 


4^ 
d 


a 

2 

H 


3 ^ 




2; 






;2; 


a! 


1 • 











































































































































































































References. 

L Compt. Rend. Acad. Sci. (Paris) (1893), 116, 1385. 

2. Exp. Sta. Rec. (1906-7), 18, 429. 

3. Exp. Sta. Rec. (1893-94), 5, 1010. 

4. Centbl. f. Bakt. 2 Abt. (1902), 9, 43-54, 107-112. 

Questions. 

1. How important is this fixation under normal conditions? 

2. What factor may be responsible for considerable fixation by 
these organisms? 

3. Compare the carbon required by Clostridia and Azotobader 
forms. 

4. Are endospores formed by these organisms? 

5. What organic acids do they produce? 

6. What sources of carbon wiU suffice for their growth? 



PRACTICE 20. 

DENITRIFICATION AND FORMATION OF 
CALCIUM CARBONATE. 

Denitrification, the reverse of nitrification, includes the 
evolution of nitrogen gas, ammonia, oxides of nitrogen and 
various other volatile nitrogen compounds from nitrates, 
nitrites, ammonium compounds, and nitrogenous organic 
compounds. It should be considered as important in 
soil studies only when conditions are brought about which 
result in the nitrogen being driven into the air. Practices 
7 and 8 represent the denitrification of nitrites and am- 
monium compounds. In its stricter sense, it means the 
reduction of nitrates with the loss of nitrogen as the gas. 

Into eight 100 cc. Erlenmeyer flasks, place the following 
solutions and materials: 

1 and 2, 90 cc. denitrification solution + 0.5 gram dex- 
trose. 

3 and 4, 90 cc. Ca(N03)2 denitrification solution + 0.5 
dextrose free from carbonate. 

5 and 6, 90 cc. base solution. 

7 and 8, 90 cc. base solution. 

Plug and sterilize at 10 pounds for 10 minutes. Inocu- 
late when cool as follows: 

1 and 2, 1 cc. fresh soil infusion (surface). 
3 and 4, 1 cc. fresh soil infusion (surface). 
5 and 6, 3 grams fresh horse manure. 
7 and 8, 5 grams rich brown silt loam. 

Pour paraffin oil over the surface and incubate at 30° C. 
for 10 days or longer if necessary. Test for nitrate, 
nitrite, and ammonia in 1-2 and observe the precipitation 

57 



58 



SOIL BIOLOGY 



of calcium carbonate in 3 and 4. Filter, dry, and test for 
carbonate by treating with acid. Allow numbers 5, 6, 7, 
and 8 to remain several weeks, after which time determine 
the loss of nitrogen by analyzing the contents of each tube 
for total nitrogen. The analyses of samples of the manure 
and soil used should also be made at the beginning. 



Date 


Sample 
number 


Treat- 
ment 


HCU 


= NH40H 


Titrated 

back, 

NH4OH 


Equiva- 
lent in 
sample, 
NH4OH 


Mgs. 


Loss 















































































































QUALITATIVE TESTS. 





1 


2 


3 


4 


5 














Nitrite 













References. 

1. Soil Conditions and Plant Growth, Russell (1913), 98-99. 

2. U. S. Dept. Agr., O. E. S. Bui. 194, 68-71. 

3. Jour. Agr. Sci. (1911-12), 4, 145-149. 

4. Science (1913), N. S., 37, 552. 

5. Science (1915), N. S., 41, 624. 

Questions. 

1. Is denitrification of importance in normal soils? 

2. In what kind of soils is it of importance? 

3. What common substances hasten denitrification? 



DENITRIFICATION 59 

4. Write a typical reaction showing denitrification, 

5. Write the reaction occurring in flasks 3 and 4. 

6. Explain the theory of the precipitation of calcium carbonate 
or ground hmestone and the formation of dolomite. 

7. Explain the physiological significance of denitrification. 

8. What other reductions are closely related to denitrification? 



PRACTICE 21. 

SULFOFICATION AND DESULFOFICATION IN 
SOILS. 

The oxidation and reduction of suKur, sulfide, sulfi,te, 
thiosulfate, and organic sulfur is a function of certain 
higher soil bacteria. The sulfur cycle like that of nitro- 
gen is extremely difficult to control as sulfur is only 
returned to the soil by rain. No organisms are known to 
fix sulfur from the air as occurs with carbon dioxid or 
nitrogen. 

Place 100 grams of soil in each of ten 400 cc. shaker 
bottles. Treat as follows : 

1 and 2, nothing. 

3 and 4, 5 cc. of sodium sulfide solution = 0.2 gram 
NasS. 

5 and 6, 0.2 gram free sulfur. 

7 and 8, 5 cc. of calcium sulfate = 0.1 gram CaS04 
(saturated). 

9 and 10, 2 grams clover tops. 

Mix the treatment thoroughly and add water to the 
optimum for all but 7 and 8, which are saturated. Plug 
the bottles and incubate at room temperature or at 30° C. 
for 14 days. At the end of that time add 300 cc. of acidi- 
fied water (HCl 5 cc. per liter), shake for 5 hours and let 
stand over night, remove 200 cc, and determine the 
sulfates as indicated on page 117. 



60 



SULFOFICATION 



61 



Group Soil Type . 

Standard Reading mm. Date . . . . 





Treat- 
ment 


Total 
volume 


Volume 
taken 


Reading, 
mm. 


Mgs. 
S 


P.P.M. 

S 

oxidized or 

reduced 





































































































































































References. 

1. Handbuch der technischen Mykologie, Lafar (1904-6), 3, 
214-244. 

2. Vorlesungen iiber landwirtschaftliche Bakteriologie, Lohnis 
(1913), 192-197. 

3. Wis. Agr. Exp. Sta. Research Bui. 14. 

4. Ken. Agr. Exp. Sta. Bui. 174 and 188. 

5. Soil Science (1916), 1, 533-539. . 

Questions. 

1. In what form is most of the sulfur in soils and what form 
do plants prefer? 

2. In what form does most of the sulfur pass into the air from 
bacterial decomposition and combustion? 

3. How are the losses of sulfur compensated? 

4. What bacteria effect the oxidation and reduction of sulfur 
and its compounds? 



62 SOIL BIOLOGY 

5. On what compounds is their action most necessary for the 
continuance of the sulfur cycle? 

6. Write the reactions involved in the oxidation of the sulfide, 
free suKur and sulfite to sulfate, the reduction of sulfate to sulfide. 

7. Write a reaction showing the reduction of sulfur by a cellulose 
decomposer; the reduction of a sulfide with the formation of calcium 
carbonate. 

8. Write the reaction showing the oxidation of tetrathionate. 



PRACTICE 22. 

FUNGI IN SOILS. 

RELATION TO SOIL NITROGEN. 

Factoes Influencing Fungous Growths in Soils. 

Weigh out fourteen 50-gram portions of the soil to be 
tested and place in the jelly glasses. 
Treat as follows : 

1 and 2, nothing, 

3 and 4, 10 milhgrams of nitrogen as nitrate. 
5 and 6, 3 grams organic matter. 
7 and 8, 3 grams organic matter (moisture 80 per cent 
of saturation). 

9 and 10, 3 grams organic matter + 10 milligrams of 
nitrogen as nitrate. 

11 and 12, 5 cc. sulfuric acid. 

13 and 14, 5 cc. sulfuric acid + 3 grams organic matter. 

The amount of water to be added will be posted on the 
bulletin board. Mix thoroughly and place in room- 
temperature incubator. Examine closely every 2 days 
for growth and finally examine a portion of the soil from 
numbers 1 and 5 with the binocular microscope for my- 
celial threads. 

At the end of 14 days analyze numbers 3 and 9 for 
nitrate, at 30 days analyze 4 and 10 for nitrate. 

Make observations on the growth in all the treatments 
after one week and then discard all except those on which 
the nitrates are to be determined. 

63 



64 



SOIL BIOLOGY 



Date 


Soil 
No. 


Treat- 
ment 


Incubation 


Growth 


Mgs. N 
as ni- 
trate 
found • 


Original 

N as 
nitrate 


Gain or 

loss of 
N as 
nitrate 


Notes 




Temp. 


Time 





































































































































































References. 

1. Household Bacteriology, Buchanan (1913), 50-84, 487-523. 

2. N. J. Agr. Exp. Sta. Bui. 270. 

3. Centbl. f. Bakt. 2 Abt. (1914), 40, 555-647. 

4. Science (1914), 39, 35-37. 



Questions. 

1. What kinds of fungi inhabit soUs? 

2. Which of these kinds predominate? 

3. Of the factors necessary for growth which are most important 
for soil fimgi? 

4. In the struggle for food how are soil fungi at an advantage? 

5. Name 10 forms of nitrogen which fungi are able to assimilate. 

6. Are fungi able to fix atmospheric nitrogen? 

7. Did the fungi feed on organic matter or nitrates in this ex- 
periment? 

8. Are fungi able to ammonify? 



PRACTICE 23. 
PROTOZOA IN SOILS. 

ISOLATION AND STUDY OF AMCEB^, CILIATES, AND 
FLAGELLATES FROM SOIL. 

Determination of Active Protozoa in Soils. 

The three classes amoebse, ciliates, and flagellates are 
commonly found in normal soils. At times they are more 
easily found owing to increases in moisture content, but 
they exist in an active state in all normal soils yet examined. 

Prepare a 1.5 per cent solution of blood meal in tap water, 
filter and add a crystal of di-potassium phosphate, and 
place 10 cc. in each of six 100 cc. Erlenmeyer flasks. Plug 
and sterilize in the autoclave at 12 pounds pressure for 
15 minutes. Treat as foUows: 

1, nothing. Inoculate, 5 grams surface soil (normal). 

2, 1 gram ground limestone, inoculate, 5 grams surface 

soil (normal). 

3, 1 gram ground limestone, inoculate, 5 grams subsoil 

(normal). 

4, 1 gram ground limestone, inoculate, 5 grams surface 

soil (poor). 

5, Make slightly acid with hydrochloric acid, 5 grams 

surface soil. 

6, 1 cc. rich algal slime, 5 grams surface soil. 

Examine each treatment at the end of two days for 
ciliates and flagellates and at intervals of two days for 
three to four laboratory periods. Note shape of these 
organisms, size, means and kind of motility, feeding 
habits and source of food. Stain some organisms from 
No. 2 with aqueous methylene blue and gentian violet 
(equal parts). Kill and stain with picrosulphuric acid. 
Carefully examine for cysts. With the experience now 

65 



66 



SOIL BIOLOGY 



acquired a direct examination for active protozoa in field 
soils may be made as follows: 

Place 100 grams of soil in a 400 cc. shaker bottle, add 
200 cc. of sterile water and 1 gram of calcium carbonate, 
shake and let settle a few minutes. Remove several 
drops from the surface with a glass rod and place them in 
a hollow cell or on an ordinary slide. Examine with the 
binocular, using the high power lens, or with the regular 
microscope, using the low power lens. Make several 
examinations. Note the forms which are motile. Study 
the plates in Dolfein, Minchin, and Bui. 2 Geological and 
Natural History Survey, Conn. 

The large forms observed are not known to possess an 
encyst stage. It should also be stated that authorities 
differ as to the time required for excystment. 

Those students desiring to make permanent slides are 
referred to advanced practice Active Protozoa in Soils. 



Sample 


Treat- 
ment 


Amoebae 


Ciliatea 


Flagellates 


number 


Large 


Small 


Large 


Small 


Large 


Small 



















PROTOZOA IN SOILS 67 

References. 

1. An Introduction to the Study of Protozoa, Minchin (1912). 

2. Conn. Geo. and Nat. His. Survey (1904-5), 1, Bui. 2. 

3. Lehrbuch der Protozoenkunde, DoKein (1911). 

4. Jour. Agr. Sci. (1909), 3, 111-144. 

5. Jour. Agr. Sci. (1915), 7, 49-74, 106-119, plates after 118. 

6. Centbl. f. Bakt. 2 Abt. (1914), 39, 596; 41, 625-630. 

7. Jour. Agr. Res. (1915), 4, 511-559; 5, 137-139; 5, 477-487. 

8. Protozoa in Illinois Soils, Reference Shelf. 

9. SoU Science (1916), 1, 135-153. 
10. Jour. Bact. (1916), 1, 423-433. 

Questions. 

1. Give the distinguishing characteristics of the three classes of 
protozoa. 

2. What constitutes the food of each class? 

3. Describe the process of encystment. 

4. What is enflagellation and exflagellation and how may they 
lead to erroneous conclusions? 

5. Give the names of typical members of each class. 

6. What factor is most important for most of the protozoa in 
soils to lead an active existence? 

7. How do you explain the fact that the protozoa may be active 
in a soil with a low moisture content? 



PRACTICE 24. 
ALG^ IN SOILS. 

RELATION OF ALG^ TO SOIL NITROGEN. 

Comparative Numbers of Alg^e in Different Soils. 

Factors Influencing the Growth of Alg^ 

in Soils. 

Place 50 grams of clean white sand in each of ten 100 cc. 
Erlenmeyer flasks. Add 20 cc. of algae solution to each, 
plug with cotton, and sterilize for 30 minutes at 12 pounds 
pressure in the autoclave. Slant the flasks so that a sand 
slope is formed. 

Prepare an infusion of the soils to be studied. In each 
of ten flasks place 10 cc. of the infusion = 5 grams of soil, 
and treat as follows: 

1 and 2, place in incubator (dark). 

3 and 4, place in window sill (direct sunlight). 

5 and 6, 0.5 gram sodium carbonate (direct sunlight). 

7 and 8, 2 cc. N/1 hydrochloric acid (direct sunlight). 

9 and 10, 6 cc. N/l hydrochloric acid (direct sunlight). 

When a green color is observed in those on the window 
sill make a microscopic examination of all the flasks. 
Note the forms and their appearance. Stain with iodine 
solution and explain the results. After several weeks, 
make stains with picronigrosin. Test all the flasks for 
nitrate. Filter the solution before testing it. 

Prepare the following dilutions of the soil infusion 
1-1000, 1-5000, 1-10,000, 1-20,000. In flask 11 place 
1 cc. of infusion 1-1000, in No. 12, 1-5000, in flask 13, 
1-10,000, in flask 14, 1-20,000. Shake the flasks and 
slant to form a sand slope. Place in direct sunUght. 

68 



ALG^ IN SOILS 



69 



Report the number of algae per gram of the soil tested. 
Counting is accomplished by observation of the green 
color and represents the minimum number. 



Date 


Soil 


No. 


Treat- 
ment 


Iodine 
test 


Nitrate 
test 


No. 

per 
grana 


No. 
per 
acre 


Notes 



















































































































































References. 

1. Chem. Absts. (1915), 9, 2923. 

2. Tufts College Studies Scientific Series 2, No. 3, The Green 
Algae of North America, CoUins, also 3, No. 2. 

3. Morphologie und Biologie der Algen, Oltmann. I SpecieUie 
Tiel. 

4. Col. Exp. Sta. Bui. 184. 

[ 5. Minnesota Algse I (Tilden). 

Problems. 

1. How many pounds of algse would it require to furnish the 
energy for a fixation of 15 poimds of atmospheric nitrogen? 

2. Calculate the nitrogen as nitrate that would be conserved per 
acre with 13^ inches of drainage from the results obtained in your 
experiment. 

Questions. 

1. In what kinds of soils do algse grow best? 

2. What recently isolated constituent of algae accounts for the 
symbiosis with Azotobacter? 



70 SOIL BIOLOGY 

3. Explain the symbiosis in view of the fact that algse require 
inorganic nitrogen and Azotobacter cannot nitrify. 

4. In what region of the soil do the algse grow? 

5. What factor is most essential for thair -existence? 

6. Explain the symbiosis between algae and paramoecium. 



PRACTICE 25. 
A STUDY OF ENZYMES. 

Enzymes are a product of living cells but after formed 
may act independently of the cell. Their exact composi- 
tion and their relation to the reactions they produce is yet 
undecided. They are likened to catalyzers, magnetism, 
and colloids and perform like all three. Two kinds are 
recognized, the endo-enzyme and the ecto-enzyme. Oxi- 
dations, reductions, hydrolytic, synthetic, and intramolec- 
ular processes are brought about by these agents. Thus 
it will be seen that the ecto-enzyme makes food soluble 
without, for assimilation by the cells and the endo- 
enzymes are responsible for digestion and fermentation 
within. 

The importance of enzyme action is better realized when 
it is stated that all our soil microorganisms perform their 
functions through these agents. 

Isolation of Proteolytic Enzyme (Proteases). 
Make 2 stab cultures of nutrient gelatin with B. subtilis, 
2 with B. liquefaciens, 2 with B. coli and 2 of Aspergillus 
niger. 

Incubate at room temperature until the gelatin is 
liquefied by B. subtilis. Add 1 cc. of toluene to each 
tube, shake, grind in an agate mortar with a small amount 
of fine sand. Filter through paper, infusorial earth, or by 
using a Berkefeld apparatus. 

Inoculate tubes of solid gelatin and milk with 5 cc. of 
the filtered product. Place a small amount of barium 
sulphate in the gelatin tubes and mark on the outside with 
a red pencil the height of the gelatin. This serves to 
show the rate and point of liquefaction. Examine each 
day and note the action which occurs. 

71 



72 SOIL BIOLOGY 

Add to a 5 per cent solution of guiacin in alcohol 5 cc. of 
the filtered product and note the results. If a color does 
not develop at once add hydrogen pe^o^iide and observe 
carefully the formation of gas or a color. 

Isolation of Urease. — (1) Inoculate 10 cc. of sterile 
urea solution with the culture obtained in practice 6. 
Incubate 24 hours at room temperature. Add 1 cc. of 
toluene to the culture and grind the solution in a mortar, 
using sand, filter and inoculate another tube of sterile 
solution with a portion of the filtered product. Add 
toluene to the tube and after 24 hours titrate the ammonia 
produced. (2) Place 0.9 gram of powdered soy bean 
seeds in 10 cc. of water and allow the mixture to stand over 
night. Separate the aqueous portion by centrifugation 
and mix the aqueous extract with an equal volume of 
glycerol. Test the soy bean urease preparation on urea 
solution. Tabulate your observations on the action of the 
different organisms on the gelatin and milk and the results 
of the chemical tests. 

References. 

1. Greneral Chemistry of Enzymes, Euler (Pope), (1912). 

2. The Nature of Enzyme Action, Bayhss (1914), 1-6, 10-13, 22, 
139-43, 146. 

3. An Introduction to the Chemistry of Plant Products, Haas 
and HiU (1913), 334-372. 

4. Bacteriological and Enzyme Chemistry, Fowler (1911), 256- 
272. 

Questions. 

1. What function does the toluene perform? 

2. Explain the production of color with and without the addition 
of hydrogen peroxide. 

3. The gas formed proves the presence of what enzyme? 

4. Classify the enzymes as endo- or ecto-enzymes. 

5. What important action does urease produce? 

6. How does the energy liberated by the two classes of enzymes 
compare? 

7. Classify the enzymes studied according to the chemical action 
produced. 



CLASS PRACTICE 1. 

GROWTH AND STUDY OF IRON BACTERIA 
OF SOILS. 

In the soil all the mineral elements are subject to direct 
or indirect bacterial action. Iron and manganese are 
typical of the elements which are attacked by special 
forms of bacteria. In studies of drinking water the 
action of these bacteria has presented important economic 
problems. These organisms occur in soils and, when suit- 
able conditions for their multiplication are brought about, 
they act on the iron and manganese compounds and 
oxidize them, thereby obtaining energy. 

In a liter Erlenmeyer flask place 500 cc. of solution for 
iron bacteria. Titrate 20 cc. of the solution for ferrous 
iron. Introduce an iron wire bent in the form of a crook 
at the end immersed and extending to the top of the flask 
bent at right angles to hold it in place. Plug and sterilize 
at 10 pounds for 15 minutes. 

Inoculate with 100 grams of fresh surface soil. Place 
the flask on the window sill and observe carefully the 
yellowish-red slime which forms on the wire and in the 
liquid. Remove some of the growth and wash the iron 
deposits rfrom the cells with 1 per cent hydrochloric acid. 
Stain with carbol-fuchsin and methylene blue. Describe 
the organisms and draw typical cells. Remove 20 cc. 
portions and determine the ferrous iron. The difference 
between the original and the final titrations represents the 
iron oxidized to ferric. 

Original titration cc. N 10 KMn04 

Final titration cc. N 10 KMn04 

cc. N 10 KMn04 mgs. Fe 
73 



74 SOIL BIOLOGY 

References. 

1. Centbl. f. Bakt. 2 Abt. (1904), 11, 215-219, 277-287. 

2. Centbl. f. Bakt. 2 Abt. (1908), 20, 97-99. 

3. Proc. Soc. Am. Bact. Pubs, in Science (1916). 

Questions. ' 

1. What influence on solubility of iron and manganese do these 
bacteria exert? 

2. Are these simple forms of bacteria? 

3. Why is the hydrochloric acid necessary? 



CLASS PRACTICE 2. 

DENITRIFICATION IN SOLUTION BY SOIL 
BACTERIA. 

This practice is suggested where laboratory facilities 
and time are limiting factors. It serves to illustrate 
denitrification, gas production, desulfofication, and ability 
of soil organisms to reduce organic compounds. 

In a large flask or bottle with a long neck (5| liter 
boiling flask) fitted with a one-hole rubber stopper, insert 
a glass tube which should extend to within I inch of the 
bottom and project 2 or more feet above the stopper. 
Bend the end of the tube so it will deliver into a graduated 
glass cylinder. Place 100 grams of surface soil in the 
flask, and fill with solution for denitrification (class prac- 
tice) so that the solution will be visible just above the 
stopper. 

Put the apparatus in some convenient place, covering all 
but the neck with a paper or cloth to shut out the light. 

The students should mark the height of the liquid on 
the tube at the beginning and every two days and record 
it on a card attached to the apparatus in inches. The 
liquid displaced by the gas should be recorded. This 
liquid may be tested for nitrates, sulfates, and sugar from 
time to time. An explanation of the process should be 
given by the instructor. 



75 



ADVANCED PRACTICE 1. 
ACTIVE PROTOZOA IN SOILS. 

The method of Martin and Lewin with careful manipu- 
lation gives excellent results. 

Place 20 grams of soil in a 100 cc. evaporating dish and 
add through a funnel at the bottom of the soil mass enough 
of the picric acid fixative or the corrosive fixative to com- 
pletely cover the soil, shake the dish immediately. Mark 
a cover shp to indicate the film side and float it on the 
surface to obtain a film. Fix, stain, and prepare the film 
for mounting by the method found on page 106. Mount 
in balsam. By means of the camera lucida draw the 
different forms observed. Stain some films with aqueous 
solution of methylene blue and gentian violet. 

Number of Protozoa in Soils. — The number of 
protozoa per gram of soil is at present best obtained by 
use of the (Blutkorperzahl apparat) blood-counting ap- 
paratus. Prepare an infusion of the soil to be examined, 
remove 10 cc, and thoroughly shake. Make a dilution 
of 1-100 or 1-10 with mixing pipettes. Discard 3 drops 
of the dilution and then place a drop in the counting 
chamber. Exercise care not to have the liquid run into 
the moat. Count the protozoa in the drop and repeat on 
3 more drops taking the average number of the four deter- 
minations and multiply by the dilution. Report the 
number per gram of soil taken. 

Separation of Protozoa. — Filter 10 cc. portions of 
stock solutions of soil protozoa through single and quad- 
ruple filters (Schleicher and SchuU's 589), which have been 
sterilized by alcohol. Count the protozoa in the filtrate 
by the method used above, and at the same time count the 

76 



ACTIVE PROTOZOA IN SOILS 77 

forms in the stock culture. After making a separation of 
this kind, prepare concentrated solutions by incubation 
and inoculate into sterile, unsterile, moist and dry soils 
and study the nitrate formation. 

Reference. 

1. Jour. Agr. Research (1915), 5, 137-139. 



ADVANCED PRACTICE 2. 

DECOMPOSITION OF CYANAMID. 

Calcium cyanamid is a product of the electric furnace. 
It is made at a high temperature (1300° C.) from calcium 
carbide and nitrogen gas obtained from the air according 
to the reaction: 

CaCa + N2 ^ CaNCN + C. 

Micro5rganisms decompose it into nitrate as shown by 
the reactions below. 

( CaNCN + CO2 + H2O = CaCOa + H2NCN. 
\ CNCaN + 2 H2O = CNNH2 + Ca(0H)2. 

2. H2NCN + H2O = CO(NH2)2. 

3. CO(NH2)2 + 2H2O = (NH4)2C03. 

4. (NH4)2C03 + 6 0+ CaCOa = Ca(N02)2 + 2 CO2 

+ 4 H2O. 

5. Ca(N02)2 + O2 = Ca(N03)2. 

The manufacture of this product has been developed to 
a point where it is cheaper than nitrate of soda and is 
destined to replace nitrate of soda in foreign countries. 

As seen from above it presents a most interesting 
chemical phenomena, a diamide (urea) resulting from 
bacterial action on calcium, carbon, and nitrogen. 

Place 100 grams of soil in each of 10 jelly glasses. Ar- 
range as follows: 

1 and 2, nothing. 
3 and 4, nothing. 
5 and 6, 0.1 gram CaCN2. 
7 and 8, 0.1 gram CaCNa. 
9 and 10, 0.2 gram CaCNg. 
78 



DECOMPOSITION OF CYANAMID . 



79 



Add moisture to the optimum as usual. 
Determine ammonia on 1, 2, 4. and 5 after 2 weeks. 
After 4 weeks determine the nitrate in 3, 4, 6, 7, 9, and 10. 



Sample 
number 


Treat- 
ment 


HCI < 


= NH4OH 


Titrated 

back, 
NH4OH 


Eqiuva- 
lent in 
sample, 
NH4OH 


Mgs. N 
as NHa 


Mgs.N 
asNOa 


1 
















2 
















3 
















4 
















5 
















6 
















7 
















8 
















9 
















10 

















Into four 500 cc. Erlenmeyer flasks place 50 cc. of the 
following solution : 

Water 1000 cc. 

Dipotassium phosphate 1.0 gram 

Asparagin 0.1 gram 

Dextrose 0.1 gram 

Calcium cyanamid 2.0 grams 

Inoculate with 10 grams of surface soil and incubate for 
4 weeks. 



80 SOIL BIOLOGY 

Prepare cyanamid gelatin by adding 10 per cent of 
gelatin, and sodium hydroxid to make faintly alkaline, 
to this solution. After 2 weeks transfer several loopfuls 
from the flasks inoculated with soil to the other two and 
after 2 weeks plate out from these solutions to the cyana- 
mid gelatin. 

Study the colony characteristics and inoculate urea 
agar and urea solution with typical colonies. 

References. 

1. Centbl. f. Bakt. 2 Abt. (1905), 14, 87, 389. 

2. Centbl. f. Bakt. 2 Abt. (1909), 22, 254-281. 

3. Centbl. f. Bakt. 2 Abt. (1910), 26, 633, 643. 

4. U. S. Dept. Com. & Labor, Bur. Mfgs. Special Agent Series 
No. 52 (1912), 116-168. 

5. Dictionary of Applied Chemistry, Thorpe (1912), 3, 698-712. 

6. lU. Agr. Expt. Sta. Bui. 179, 494, footnote. 

Question. 

1. Discuss the importance of the cyanamid industry as related 
to general agriculture. 



ADVANCED PRACTICE 3. 
PROTEIN FORMATION IN SOILS. 

This practice serves to demonstrate that certain bacteria 
use nitrate nitrogen to make protein instead of evolving 
the nitrogen gas into the air. It tends to show that these 
organisms with normal moisture content may act as con- 
servers of nitrogen instead of denitrifiers. 

Place 100 grams of soil in each of ten 500 cc. Erlenmeyer 
flasks. Sterilize six of them at 15 pounds pressure for 
2 hours. 

Inoculate and treat as follows: 

No. 1. Normal soil, nothing, 18 per cent water. 

No. 2. Normal soil, 0.5 gram dextrose, 18 per cent water. 

No. 3. Normal soil, 0.5 gram dextrose, 18 per cent water + 10 mga. 

N as nitrate. 
No. 4. Normal soil, 0.5 gram dextrose, 18 per cent water, + 50 mgs. 

N as nitrate. 
No. 5. Sterilized soil, 0.5 gram dextrose, 18 per cent water, 10 mgs. 

N as nitrate. 
No. 6. Sterilized soil, 0.5 gram dextrose, 24 per cent water, 10 mgs. 

N as nitrate. 
No. 7. Sterilized soil, 0.5 gram dextrose, 30 per cent water, 10 mgs. 

N as nitrate. 
No. 8. Sterilized soil, 3 grams dextrose, 18 per cent water, 50 mgs. 

N as nitrate. 
No. 9. Sterilized soil, 0.5 gram dextrose, 24 per cent water, 50 mgs. 

N as nitrate. 
No. 10. Sterilized soil, 0.5 gram dextrose, 18 per cent water, 100 mgs. 

N as nitrate. 

The sterilized treatments are inoculated with cultures 
of B. fluorescens liquefaciens and B. pyocyaneus. Total 
nitrogen determination should be made on the soil and 
added materials in the beginning. 

' 81 



82 



SOIL BIOLOGY 



The sugar and nitrate are added, together with the 
inoculation, in the sterile water applied. 

Incubate at room temperature for . 3-4 weeks, after 
which time, dry and grind the soil and determine total 
nitrogen in duplicate in samples of ten grams. Determine 
the nitrate nitrogen in samples of 50 grams. 



Num- 
ber 


Treat- 
ment 


Total nitrogen 


Nitrate nitrogen 


Protein 


Found 


Mgs. 
calcu- 
lated 


Gain -|- 

or 
loss — 


Found 


Mgs. 
calcu- 
lated 


Gain -|- 

or 
loss — 


N, mgs. 
formed 























































































































































































References. 

L Centbl. f. Bakt. 2 Abt. (1910), 26, 335-345. 

2. Soil Conditions and Plant Growth, Russell (1913), 99. 



Question. 

1. Explain the results obtained in terms of the conservation of 
nitrogen. 



ADVANCED PRACTICE 4. 

FLAGELLA STAINING OF B. RADICICOLA, B. 

NITROSOMONAS, B. DENITRIFICANS, B. SUB- 

TILIS, B. TYPHOSUS. 

Some of the common and more easily stained organisms 
are used for comparison with the more difficult, such as 
B. radicicola. This is a difficult procedure and requires 
careful technique. The organisms to be stained should 
have been transferred every two days for several times 
and before staining examined in the hanging drop. It is 
best to have transfers of each organism as some are better 
than others. The cover slips must be absolutely clean 
(test with water). After removing from the alcohol, if 
suspicious, pass through the flame three times allowing time 
between each to avoid cracking. Place 1 loopful of water 
on the cover shp. Spread by pulling and do it gently. 
Care at this point is highly essential. The end of the 
platinum wire may be used for this purpose. First fix 
with gentle heat and then apply the mordant (Loeffler's 
Flagella Stain) which should be filtered on to the cover 
slip until completely flooded and heat until steam arises 
(use care not to overheat). Hold the cover slip with 
cornet forceps and keep the finger extended over it when 
passing through the flame. Heat |-1 minute. Wash in 
water and blot with fllter paper. Stain with the carbol- 
fuchsin, taking care to completely flood the cover slip so 
that no material will be precipitated out during the 3 
minutes heating. Wash and examine. Mount in balsam 
if a satisfactory stain is obtained. Keep accurate notes 
on the procedure. 



83 



ADVANCED PRACTICE 5. 

CROSS INOCULATION OF LEGUMES. 

Some valuable cross inoculations have already been 
made by this station. There are no doubt many other 
similar cross inoculations which may be made. The 
following method will serve to enable advanced students 
to test some of these possibilities. Sterilize clean white 
sand in 600 cc. beakers. Add 5 cc. sterile plant food 
solution (nitrogen omitted) in 140 cc. of sterile water. 
Sterihze the legume seeds in 1-500 mercuric chloride solu- 
tion and with a sterile pipette add the legume bacteria in 
the following experiment : 

1- 5, 1st legume, inoculated with bacteria common to it. 

6-10, 2nd legume, inoculated with bacteria of legume No. 1. 
11-15, 2nd legume, inoculated with bacteria common to it. 
16-20, 1st legume, inoculated with bacteria of legume No. 2. 
21-25, 1st legume, uninoculated (sterile). 
26-30, 2nd legume, iminoculated (sterile). 

Failure to prove out organisms in some such manner at 
the conclusion of experiments may lead to erroneous work. 

There are many wild legumes which should be tested 
on the cultivated. Wild vetches, beans, peas, lespedeza, 
desmodium (ticks), beggarweed, coffeeweed and many 
more and their relation to beans (common), soybeans, 
and many others are typical cases which are not yet 
solved. 

If organisms are not available, gather soil about the wild 
legumes and grow the nodules with which to start. Some 
of the crosses made by earlier workers should be repeated 
as only a few are known to be reliable. The plants are 
allowed to grow in the greenhouse. If a suitable undis- 

84 



CROSS INOCULATION 85 

turbed portion of a greenhouse is available, it is not 
necessary to keep the plants under sterile conditions. 
Insects and people should be excluded. Separating the 
treatments is a further precaution. 

References. 

1. lU. Agr. Exp. Sta. Bui. 179, 497-498, 475. 

2. Manual of Botany, Gray, 7th Edition (1908), 499-531. 

3. Illustrated Flora of the Northern States and Canada, Britton 
and Brown (1913), II Amaranth-Logania, 330-425. 

Questions. 

1. How closely related were the orders crossed? 

2. Discuss the possibility of these organisms growing on plants 
not legumes. 



ADVANCED PRACTICE 6. 

SOLVENT ACTION OF SOIL BACTERIA ON 
MINERALS. 

SOLUBLE PHOSPHORUS AND CALCIUM PRODUCED BY 
NITROSOMONAS. 

The formation of nitrites requires a base and the calcium 
of insoluble phosphates is a suitable one. This results in 
a liberation of soluble phosphorus. 

Place 50 cc. of base solution for nitrite formation in each 
of eight 1 liter Erlenmeyer flasks, plug loosely and sterilize 
in the autoclave at 10 pounds pressure for 15 minutes. 
Add 20 milligrams of nitrogen as ammonium sulfate and 
100 milligrams of the raw rock phosphate to each flask. 
The phosphate should have been dried and analyzed for 
phosphorus and calcium before using. 

Arrange as follows: 

1 and 2, sterile rock phosphate.* 
3 and 4, inoculated rock phosphate. 
5 and 6, inoculated rock phosphate. 
7 and 8, inoculated rock phosphate. 

Inoculate each with 5 cc. of a fresh infusion of soil and 
then again sterilize flasks 1 and 2. Place in 30° incubator 
and at the end of 40 days proceed as follows: Filter the 
contents of the duplicate flasks 3 and 4 through an S. and S. 
589 and make up to 200 cc. Analyze two 50 cc. for nitro- 
gen as nitrite and nitrate and two 25 cc. portions of phos- 
phorus and calcium. Use the Devarda method for the 
nitrogen and the volumetric methods for phosphorus and 

* Pure tricalcium phosphate and various kinds of insoluble 
natural phosphates are used in this experiment. 



SOIL BACTERIA 



87 



calcium. After 50 days analyze flasks 5 and 6 and after 
60 days flasks 1, 2, 7, and 8. Calculate the ratio of nitro- 
gen oxidized to phosphorus and calcium made soluble. 



Num- 


Nitrogen 
oxidized 


Phosphorus 
soluble 


Calcium 
soluble 


Ratios 


ber 


N:P 


N:Ca 


Ca:P 






- 











References. 

1. ni. Agr. E^. Sta. Bui. 190. 

2. Centbl. f. Bakt. 2 Abt. (1904), 11, 724. 

3. SoU Science (1916), 1, 533-539. 

Problems. 

1. Write the chemical reaction to which the resnlta conform. 

2. Calculate the possible amounts of phosphorus available for 
standard crops of com, wheat, oats from the reaction occurring. 



Questions. 



1. What organisms exert a solvent action on minerals? 

2. Of these which are the most important? 



88 SOIL BIOLOGY 

3. Upon what will the availability of the raw rock phosphate 
depend? 

4. Explain why the nitrite organisms are effective in this con- 
nection and the nitrate not. 

5. Discuss the importance of the plant in an equihbrium reaction 
of this kind. 



PART n. 
METHODS IN SOIL BIOLOGY. 

1. Bacteriological 91 

2. Chemical Ill 

3. Mechanical 121 

4. Pot Culture 131 

5. Suggestions for Instructors and 

Students Preparing to Teach. . . 136 



BACTERIOLOGICAL METHODS. 
FOOD OF SOIL MICROORGANISMS. 

Soil microorganisms consisting of bacteria, fungi, algse, 
protozoa, and others require certain elements for growth 
and for energy. Ten elements are essential for vegetable 
microorganisms while animal microorganisms require the 
same elements with the addition of sodium and chlorine. 

The twelve essential elements are carbon, hydrogen^ 
oxygen, phosphorus, potassium, nitrogen, sulfur, calcium, 
iron, magnesium, sodium, and chlorine. Manganese is 
associated with many microorganisms, but is not known 
to be an essential element. Silicon is thought by some to 
be necessary for diatoms. 

Vegetable microorganisms require the same ten essential 
elements as green plants, and animal microorganisms 
require the same twelve as the higher animals. In this 
respect there is a great similarity, but when the ability of 
certain microorganisms to utilize these elements from di- 
verse sources is considered there appears exceptional 
differences. 

Noteworthy among them may be mentioned the follow- 
ing: First, the utilization of free nitrogen by nitrogen 
fixers {B.radicicola, B. Clostridium and Azotobacter) as 
a source of food; second, the acceptance of either inorganic 
or organic carbon (carbon dioxide, bicarbonate or sugar) 
as a source of food by -nitrifying and sulfur organisms 
(Nitrosomonas, Nitrohacter, and sulfur bacteria); third, 
the appropriation of mineral compounds for the production 
of energy (ammonia, nitrite, hyposulfite, sulfur, iron, 
manganese, and even elementary carbon are used by 
various bacteria) ; fourth, the utilization of organic nitrog- 

91 



92 SOIL BIOLOGY 

enous compounds (urea and amino acids) as a source of 
both energy and food by urea and other bacteria. 

Other rare exceptions to the general sources of the 
essential elements are, methane which serves as food for 
growth and energy, and hydrogen gas which serves as 
food for growth only, for a few bacteria. 

It is well to recall that of the non-nitrogenous com- 
pounds, alcohols (simple and complex), carbohydrates 
(sugar, starch, and cellulose), organic acids, fats, and as 
recently shown even benzene, carbolic acid, paraffin, and 
formaldehyde are decomposed by bacteria and used as 
food for growth and energy. Among the nitrogen com- 
pounds bacteria find a source of food for growth and for 
energy and here they exhibit great variations compared 
with plants and animals. Bacteria utilize free nitrogen, 
nitrate, nitrite, ammonia, urea, amino acids, primary and 
secondary derivatives of protein (proteans and peptone), 
and protein. It is claimed there is no organic compound 
which cannot be decomposed by some bacteria. 

Plants and animals would soon become extinct if forced 
to depend upon the nitrogen of the atmosphere. Legumes, 
by symbiosis with B. radicicola, gain nitrogen coming from 
the air, but without this association they cannot use 
atmospheric nitrogen. Plants depend upon inorganic 
substances for their elements. Nitrates, ammonia, and 
possibly some nitrogenous organic compounds constitute 
their nitrogen sources of food. Animals require proteins, 
peptones, and some may assimilate amino acids. 

Bacteria, fungi, and yeasts possessing no photo-synthetic 
process as green plants do, obtain their energy chiefly by 
oxidation or intramolecular changes, or a breaking down 
of compounds (destructive metabolism). In doing this, 
they oxidize enormous amounts of material for energy 
compared with small amounts taken for cell growth. 
Plants reverse this process, requiring large amounts of 
food for growth and obtain their energy from the radiant 



FOOD OF SOIL MICROORGANISMS 93 

energy of the sun at the same time building up com- 
pounds (constructive metabolism). 

The food for energy is an important practical considera- 
tion in dealing with microorganisms especially such as 
Azotobacter or B. radidcola. It is valuable to recognize 
the source of energy of our soil organisms and supply the 
form necessary for the desirable organisms. This phase 
of nutrition has not as yet received much investigation. 

An understanding of the sources and the changes 
through which the essential elements for microorganisms 
pass is necessary, before their role in permanent agriculture 
can be appreciated. 

To this end, it has become necessary to develop many 
kinds of media which serves to enable the soil biologist to 
acquaint himseK with the characteristics of these organ- 
isms. 

PREPARATION OF CULTURE MEDIA. 

In considering culture media, it must be stated that 
logically and ultimately there is only one kind of culture 
media and that is the soil. For soil biological studies to 
have their proper relation to soil fertility and to aid in 
developing better systems of agriculture, they must be 
conducted ultimately with the natural medium of the soil. 
The more scientific research advances along these lines, 
the more convincing the above statement becomes. 

It should now be said, however, that the soil biologist 
has need^ of other culture media than soils. One must 
admit that most of our important discoveries would have 
been impossible had soil boen employed solely as the 
culture medium. A simpler medium, than a complex 
mixture such as a soil, is necessary in order to establish the 
biochemical reactions brought about and to study the 
characteristics of the organisms, 

These being established, application to the soil then is of 
greatest importance. This is not always easily done and 



94 SOIL BIOLOGY 

often requires a great amount of difficult research before 
it is possible to prove that a similar reaction occurs in soil 
and before its relative importance is understood. We 
therefore apply food for microorganisms to liquid and solid 
materials of various kinds, such as water, agar, and gela- 
tin, and as a result we have the so-called liquid and solid 
media in or on which the organisms grow. 

For convenience in use the following tabular method of 
presenting the formulae of the various culture media has 
been adopted. 



LIQUID MEDIA 



O 

Si 

O 



^ 



o-g S o 
ll §1 



".2 



^g 












I 


















o 


do 











«M : : : : : : :S^.§gS 



oo o t3 ? 

^ :::::::< ^ 



■= 3-e P-E 2^3 



" ™+i O (BT3 -d <» 

OO oj3 > c • e b 

oo c3+i ^ 3 ® ^;^ 

cort -g (3 o o 3 



-Sag 

-SgM-g 

03 — 



o 2 

- ^ p § 



o "d-g-S 
■^ -' 3 g^ 









?^s 



— ' o'ffl °"^ a 



c3 o. s <" s a 

j30fl00pc!M3.; 

"ii a a. a g fcDG=;j 



3 S a 



o §•£3 SS5-3 3.15 g rt3 ^ 



3 o S '- ■" 

j"H;a^;a h'd-o^ 



96 



SOIL BIOLOGY 



W g 

a g 
o o 
t— I ~-^ 

H 

P 

o 

CO 

O 

o 



OW 03 
■ - bO C S 

■2 g.2-S 



^■^ o tio.2 



OJ3 

2: 



9'3« 



o 
o 




















o 






- 




o 









= o.2Sc 

>}-S -O to o 
? "5 o p '^S 

12; "^ C« 











bO 
C3 

g 
















>1 














1 










d 



























lO 


- 




d 


S 

T3 





o o o 

fcH H t- 

cccoco 



o :3 



bC (£ 



d S d g 



S S c S £ ., 



mo 



■?g 



S ° " 



m:^ 



Oj? 



9 S i S «J--'3 

■2|S-g'igs§ 

• ^ +^ I o ra 2 ^ 

o o 5 fl ho rs.ri 
oj.i o E ca n! nla 



g S o g S fl 

t-l F^ .. Ih l-l i-l 

3.3.3 3.3 3 

o<^<» o<^ o 



s 



3 • s-^ 
g'^-2 g 

S OS S-3 

3 ,^ O 03 

(iHOWPt, 



SOLID MEDIA (AGAR) 



97 



p4 
< 

o 

g 

o 

03 

o 



Pi 
o 





Starch. 
500 cc. 


'.o '■ \ 




■ :i ! : i : : i° : : ! 


; uj ::::'.: ITS j 

:-(d : : : : : -^ : 


- ; 






a 
_o 

1 
o 

m 


'.•SB i ; : i : i : : i : i : : : : 








d 
o 

m 


• ■ 6 

■ ■ « 

: :§ 

■ -o 










Fungi 
gelatin. 

1000 cc. 




: Iff, ; : ■ : :=^ 

; lo !(M j • ; ;d 






1 


1 

1 
1 


8 : : : 

1" : : : 


: : : ! '. • : • .(M • • 




J 


1 


•1 ^ 

CO 


■in ■ ■ ■ 


"5 • : : : : : i"^ : : 




» 




-i 
.2'3 s 
M g. 


l\s\\ 


: : : : : : : : : -u^c 


H . -o • ■ ■ -o g o ■- 

., ; : ; ; ; : T3 : 
3 : m : : 




- 


.1 
1 

o 
O 

1 


Bouillon 


d 
2 

o 
o 

o 

3- 
"S" 


Peptone. . ._ 

Starch solution 

Dextrose 

Maltose 

Mannite solution 

Mono-potassium phosphate 


Mono-ammonium pnospnate 

Magnesium ammonium phosphate 

Ammonium sulfate 

Magnesium sulfate 

Potassium sulfate 

Sodium nitrate 

Ammonium nitrate 

Sodium nitrite 

Ferric chloride 

Sodium chloride 

Calcium carbonate 

Sodium asparaginate 





98 



SOIL BIOLOGY 



» 






So 



m S O 

lis 



.2.2 1 



. o o 
;co CO 






o o S a 

ho O 0) ? ©, 
<JPQOOPHt 



o o 

O cS 



<Bj3 D. 



S^ 



2s3^>a 



:2S-: 

^.2 









qacoocoflc 

ca S3 ca.,i< o.,i o o3 B 

iggSPSQSg«J 



3.2c"|oa°i 

sgsSog.gsa 



gpH 






SPECIAL MEDIA 99 

SPECIAL MEDIA. 

Silica Jelly. — This solid medium possesses the ad- 
vantage of being exceptionally selective for the growth of 
the nitrite and nitrate organisms. Its successful prepara- 
tion and use require careful observance of the following 
details. 

Procedure. — Standardize a sufficient amount of a solu- 
tion of sodium silicate to last several years. This is easily 
accomplished by adding hydrochloric acid, filter, wash, 
dry, and weigh as silicic anhydride (Si02). Stock solu- 
tions containing 4-5 per cent of silicic anhydride are pre- 
pared from this solution as desired. 

Prepare a 4-5 per cent solution of silicic anhydride in 
water and a standard solution of hydrochloric acid of 
equivalent strength, using methyl orange as the indicator. 
This (4-5 per cent silicic anhydride) stock solution of 
sodium silicate will keep indefinitely. When it is desired 
to prepare the jelly for plating proceed as follows: 

Solution I. 

106 cc. standard hydrochloric acid. 
Add the following salts to the acid. 
Poxu" the sodium silicate solution in the acid. 
100 cc. sodium silicate solution (4-5 per cent silicic anhydride). 
200 milligrams di-potassium phosphate. 
100 milligrams calcium chloride. 
40 milligrams magnesium sulfate. 
1 drop ferric chloride. 

Add methyl orange as indicator and sterilize in the auto- 
clave at 12 pounds pressure for 15 minutes. Dilute two 
10 cc. portions to 100 cc. and titrate against solution II, 
III, or IV (sodium carbonate solution 7.5 grams per liter), 
to determine the amount necessary to solidify the jelly. 
One cc. only should be added and if more is required, add 
more carbonate to the solution. 



100 SOIL BIOLOGY 

Solution I should not stand more than 5-6 days before 
using. It is best used immediately. 

Solution II For nitrite organisms 

Sodium carbonate 7.5 grams per liter 

Ammonium sulfate. ... 10 grams per liter 

Solution III For nitrate organisms 

Sodium carbonate 7.5 grams per liter 

Sodium nitrite 10 grams per liter 

Solution IV For nitrite and nitrate or- 
ganisms 

Sodium carbonate 7.5 grams per liter 

Ammonium sulfate .... 5 grams per liter 
Sodium nitrite 5 grams per liter 

Solutions II, III, IV, when it is necessary to sterilize 
them, should not be autoclaved, but the dry salts should 
be placed in sterile water in sterile flasks. 

Pour plates as below: 

I. Place 1 cc. of the solution to be plated in the sterile 

Petri dish. 
II. Add 10 cc. of Solution I and thoroughly mix. 
III. Add 1 cc. of Solution II, III, or IV as desired and 
tilt to mix. 

The jelly solidifies rapidly, changing in color from red to 
pale yellow, and care must be exercised to keep the plates 
level. Place in the tin boxes and incubate at 28° and 
22° C. 

Transferring the organisms from the silica jelly to 
ammonium, magnesium, phosphate agar, and nitrite agar 
has been found very advantageous, as they grow more 
rapidly on these media than on the silica jelly, but for 
first plating the silica jelly is unexcelled. 

Magnesium Plaster of Paris Blocks. — These 
blocks are sometimes used for the growth of nitrite, ni- 
trate, and nitrogen fixing organisms. 



SPECIAL, MEDIA 101 

Procedure. — Thoroughly mix 1 gram of magnesium 
carbonate with 200 grams of plaster of Paris and slowly 
add water until the mass becomes pasty. By means of 
a spatula spread the mixture on a piece of glass and mark 
into the desired shape (squares are very convenient for 
use in Petri dishes). When dry they may be pried loose 
and stored indefinitely. 

When desired, place in Petri dishes or other receptacles 
and pour in enough of the nitrite, nitrate, or nitrogen 
fixation solution to half submerge the block. Sterilize 
in the autoclave at 10 pounds pressure for 10 minutes. 
Inoculate with 1 cc. of infusion or 3 loopfuls of medium. 

Cellulose Solution (Scales Method) . — Dilute 100 
cc. of concentrated sulfuric acid with 60 cc. of distilled 
water in a 2 liter Erlenmeyer flask. Keep the acid at 
60° C. Moisten 5 grains of white ribbon S. & S. filter paper 
with water and add to the acid. Agitate until the paper 
is dissolved and quickly fill the flask with cold tap water. 
Do not take over 1 minute for this operation. Filter the 
precipitate and wash free of acid and then make up to 
500 cc. with water. To prepare a solution for anaerobic 
studies or the agar consult the table of formulae for solid 
and liquid media. 

Reaction of Cultuhe Media. — When it is desired 
to bring the medium to a definite degree of acidity or 
alkalinity, proceed as follows: 

Place 5 cc. of the solution in an evaporating dish or 
casserole. Add 45 cc. of water and boil 1 minute. Add 
1 cc. of phenolphthalein, and then run in from a burette 
A7'/20 NaOH or HCl until a faint but stable pink or no 
color with HCl remains after 1 minute of boiling. Make 
several titrations and average the results. The formula, 

cc. iV/20 NaOH or HCl used , 
20 • ^■•^- 1000 cc, 

makes the calculation of the number of cc. of normal 



102 SOIL BIOLOGY 

HCl or NaOH necessary to be added to neutralize one 
liter a simple matter. 

X is the number of cc. of normal alkali or acid required 
to exactly neutralize one liter of medium. The amount 
of alkali or acid actually added to any medium to give a 
definite degree of alkalinity or acidity is easily calculated 
as below. 

One cc, of normal alkali or acid per liter is designated 
as +1° for the acid and —1° for the alkali on Fuller's 
scale. Neutral solutions are indicated as on this scale. 

Example. — Prepare a medium +8° on Fuller's scale 
and one —8°. 

When the initial titration was made with NaOH then 
X — 8 = number cc. normal alkali to add to give +8°. 
X -\- 8 = number cc. normal alkali to add to give —8°. 
When the initial titration was made with HCl, then 
X -\- 8 = number cc. normal acid to add to give +8°. 
X — 8 = number cc. normal acid to add to give —8°. 

The erroneous use of per cent of acidity or alkalinity 
in this connection is omitted here. If the student meets 
this method in the literature, he should consider only 
degrees + or — as they are cc. of normal acids or alkalies 
per liter and from them correct calculations may be made. 
Per cent cannot be relied upon as indicative of normality 
or hydrogen ion concentration. 

The student and instructors as well should bear in mind 
that true acidity means hydrogen ion concentration. In 
adjusting the reactions of solutions where accuracy is 
desired, they had best be titrated cold or after sterilizing 
as some of the constituents of certain solutions react and 
hydrogen is lost. 

Many of the culture media outlined in this manual 
require no adjustment of the reaction. In most experi- 
ments for soil bacteria when it becomes necessary to adjust 
the medium, +8° is commonly used. 



STAINING AND PREPARATION OF STAINS 103 

STAINING AND PREPARATION OF STAINS. 

Simple Staining. 

In general, the simple stains are most satisfactory in 
soil biological studies. For simple staining the procedure 
below should be followed: 

1. By means of clean cornet forceps pass the clean slide 
through the flame three times to remove the alcohol. 

2. By means of a sterile platinum loop place a small 
drop of sterile water in the center of the slide; this serves 
to give a better spreading of organisms and to indicate to 
the student if his slide is clean. With a sterile loop place 
the bacterial substance in the water and spread. 

3. Air dry and fix by passing through the flame 3 times, 
film side up. With the forefinger at the tip of the forceps 
and describing a circle about 1 foot in diameter one round 
per second there is no danger of overheating. 

4. Flood with the stain desired. Heating is permissible 
if carefully done and sometimes is a great advantage. 
Allow Loeffler's methylene blue and gentian violet to act 
3 minutes, carbol-fuchsin 30 seconds, iodine 5 minutes, 
Lugol's iodine 3 minutes, picro-sulfuric acid aigrosin 24 
hours, Bismarck brown 3 minutes. 

5. Wash with distilled water, taking care not to injure 
the film. 

6. Dry and examine with the oil immersion lens. 

7. For permanent mounts the stain should be made on 
the cover ,slip and mounted in Canada balsam properly 
labeled, with date, name of student, and organism. 

Special Staining. 

Without the aid of special stains the presence and study 
of flagella, capsules, spores, and other characteristics 
would be unknown. 

Many of these methods require extreme care and 
patience to obtain successful results. 



104 SOIL BIOLOGY 

' Flagella Stain. 

The staining of flagella is perhaps the most difficult 
method encountered by the soil biological student. At 
times it is easy to stain flagella and at other times difficult. 
The cultures should be repeated transfers of young and 
moist colonies from agar, not over 48 hours old and should 
be examined in the hanging drop first to see if they are 
motile. Proceed as in simple staining until after spread- 
ing the bacteria, when they should be allowed to diffuse 
one-half hour; then spread with platinum needle and fix 
by gentle heating. Apply the mordant by filtering it 
upon the cover glass, warm the cover glass, allow mordant . 
to act 4-6 minutes, wash with water and apply stain im^ 
mediately, allow warm stain to act for 3 minutes. Wash 
with water, dry, and examine. 

Loeffler's Flagella Stain. 

1. Mordant (warm, allow to act 4-6 minutes). 

Solution of tannin (20 per cent in water), 10 cc. 
Saturated cold aqueous solution of ferrous sulfate. 
Remove iron oxide by filtering. Use pure crystals, 5 cc. 

2. Stain carbol-fuchsin (warm, allow to act 3 minutes). 

Spore Stain. 

(Hansen's Spore Stain.) 

It is sometimes desirable to stain for spores (endospores) 
and for this purpose, make a simple stain first which will 
partially demonstrate the presence of spores. Fix as usual 
and wash with chloroform to remove fat. Wash with 
water and dry. Fix again and then drop on carbol- 
fuchsin and heat 5 minutes over a flame, renewing the 
stain as it boils away. Nearly decolorize in dilute acetic 
acid (5 per cent). Wash and counterstain with dilute 
aqueous methylene blue or Loeffler's methylene blue. 
Wash, air dry, and examine. 



STAINING AND PREPARATION OF STAINS 105 

Capsule Stain. 

In selecting bacteria for capsule staining a viscid growth 
on agar or a slimy appearance on the surface of a solution 
suggest the presence of capsulated forms. The capsules 
do not take up the simple stains sufficiently strong owing 
to their mucilaginous nature. The capsules surround the 
cell wall and when several capsules are united the mass is 
the so-called zooglea. 

Hiss Capsxjle Stain. 

' Spread film, dry, and fix; then stain with a 5 per cent 
solution of gentian violet and steam slightly, wash at once 
with 20 per cent copper sulfate solution, dry between 
filters, and examine. 

Nodule Tissue Stain. 

(Flemming's Triple Stain.) 

This method of stain is especially adapted to differen- 
tiating tissues in the nodule (Microtome sections). 
The section is first placed in: 

(1) Safranin (sat. al. solution) 50 cc. 

Distilled water 50 cc. 

Aniline water 5 cc. 

After washing in water, it then goes into (2) : 

(2) Saturated aqueous solution of gentian violet. 

It is then washed in water and passed into (3) : 

(3) Aqueous solution orange G. or other suitable stain, 

strong or weak (about one-half saturated). 

Protozoa Fixative, Stain, and Mount. 

(Martin and Lewin Method.) 

Place soil in some receptacle which will give a large 
ratio of depth to diameter and add through a funnel to 
the bottom' of the soil layer enough of picric fixative to 
cover the soil and shake disk immediately. 



106 SOIL BIOLOGY 

Picric Fixative. 

Picric acid, 1 per cent 100 cc. 

Alcohol, 70 per cent 100 cc. 

Float cover slips marked to indicate film side on the 
surface to obtain film. Stain and mount by the method 
below: Films may be obtained by the corrosive fixative. 

Mercuric chloride (sat. solution) 100 cc. 

Alcohol, 70 per cent 100 cc. 

Treat picric films as indicated below and corrosive films 
likewise, omitting No. 1 : 

1. Corrosive solution 2 minutes 

2. Alcohol, 70 per cent + iodine in potas- 

sium iodide 5 minutes 

3. Water, distilled. 
[ hsematoxylin 1 gram 

water, distilled 1000 cc. 

sodium iodate 0.2 gram 

alum 50 grams, 5 minutes 

alum 1-2 grams, 3 times 

water 100 grams 

6. Tap water (till blue) . 

7. Alcohol, 70 per cent 5 minutes 

8. Eosin in absolute alcohol 3-5 minutes 

9. Absolute alcohol I 1 minute 

10. Absolute alcohol II 1 minute 

11. Xylol I 2 minutes 

12. Xylol II 1 minute 

Mount in balsam and preserve to be used to demon- 
strate the presence of active protozoa in normal field 
soils. 

Protozoa Stain. 

The use of certain stains serves to emphasize some of 
the functions within the cell and to differentiate the 
structures. 

Neutral red (1-800 physiological salt solution or tap 
water) distinguishes nucleus, nutrition vacuoles (alkaline 



4. Hsemalum a 



5. Wash with b 



STAINING AND PREPARATION OF STAINS 107 

yellowish red), acid fermentation granules, and fatty 
granules. 

Neutral violet colors the metachromosomes. Bismarck 
brown (1-20,000-30,000) colors the nutrition vacuoles. 
Congo red proves acid in the nutrition vacuoles. Litmus 
and ahzarin suKate also prove this. Vesuvin stains bac- 
teria in the nutrition vacuoles of protozoa a very brown 
color. 

Methylene blue and gentian violet in equal parts in 
aqueous solution are useful for distinguishing protozoa. 

Alg^ Fixative and Stain. 
(Picro-nigrosin . ) 
This stain has the advantage of fixing the organism and 
staining at the same time. It is useful in collecting 
specimens on that account. It should be allowed to act 
24 hours. Combine in equal parts the aqueous solutions 
of picric acid and nigrosin. 

FORMUL.^ OF STAINS. 

It is of advantage to have certain stock solutions of 
the stains and of other materials which do not deteriorate 
and which are used in large amounts. 

FoKMULuE OF Stock Solutions of Simple Stains. 

Methylene blue 7 grams 

Alcohol, 95 per cent 100 cc. 

Gentian violet 8 grams 

Alcohol, 95 per cent 100 cc. 

Fuchsin 4 grams 

Alcohol, 95 per cent 100 cc. 

Aniline 10 cc. 

DistUled water ; 500 cc. 

The stock solutions are best made by the application, 
of heat. The aniline should be shaken for 10 minutes and 
filtered through filter paper (this solution will not keep 
over a week). 



108 SOIL BIOLOGY 



Simple Stains. 

The simple stains are prepared as follows: It is always 
best to filter the stock solution. 

1. Methylene blue: 

Saturated alcoholic solution methylene 

blue 30 cc. 

Potassium hydroxide, 0.01 per cent solution 100 cc. 

2. Carbol-f uchsin : 

Satm-ated alcoholic solution fuchsin 5 cc. 

Carbolic acid, 5 per cent solution 45 cc. 

3. Aniline gentian violet: 

Aniline solution 50 cc. 

Saturated solution gentian violet 8 cc. 

4. Lugol's iodine solution: 

Iodine , 1 gram 

Potassium iodide 2 grams 

Water, distilled 300 cc. 

5. Iodine solution: 

Iodine 3 grams 

Alcohol, 70 per cent 100 cc. 

6. Picro-sulfuric acid: 

Picric acid 2.5 grams 

Sulfuric acid, cone 5 cc. 

Water, distilled 1000 cc. 

7. Picric acid, alcoholic: 

Picric acid 10 . grams 

Water 1000 cc. 

Alcohol, 70 per cent 1000 cc. 

8. Nigrosin, alcoholic: 

Nigrosin 1 gram 

Alcohol 196 cc. 

Water 4 cc. 

9. Nigrosin, aqueous: 

Nigrosin 2 grams 

Water, distilled 1000 cc, 

10. Bismarck brown: 

Bismarck brown 2 grams 

Alcohol, 70 per cent 100 cc. 

11. Fuchsin, aqueous: 

Fuchsin 1 gram 

Water 100 cc. 



STAINING AND PREPARATION OF STAINS 109 



FoEMUKE OP Stock Solutions op Disinfectants. 

1. Carbolic acid 50 grama 

Water, distilled 1000 cc. 

2. Carbolic acid 20 grams 

Alcohol 100 cc. 

3. KOH (sticks) 1 . 25 grama 

Water, distilled 1000 cc. 

4. Corrosive sublimate, HgCl2 sat. in water.. . . 1000 cc. 
Alcohol, 70 per cent : 1000 cc. 

5. Corrosive subUmate, HgCU 1 gram 

Water 300 cc. 

6. Corrosive sublimate, HgCl2 1 gram 

Water 500 cc. 

7. Corrosive subUmate, HgCU 1 gram 

Water 1000 cc. 

8. FormaUn alcohol: 

Commercial formalin (40 per cent formal- 
dehyde) 2 cc. 

Alcohol, 70 per cent 100 cc. 

9. Formalin aqueous: 

Formalin 2 cc. 

Water, distilled 98 cc. 

10. Formalin strong for preserving specimens: 

Formalin 4 cc. 

Water, distilled 100 cc. 

Special Stains. 

1. Orange G.: 

Orange G. 1 gram 

Water, distilled 100 cc. 

2. Safranin : 

Safranin 1 gram 

Alcohol, 95 per cent 50 cc. 

Water, distilled 50 cc. 

3. HsematoxyUn: 

Saturated solution arnmonia alum 100 cc. 

Add drop by drop solution of hsematoxylin 

6 cc. alcohol 1 gram 

Expose to air one week. FUter and add 25 

cc. 'glycerin and 25 cc. methyl alcohol. 

Let age 2 months before using. 



110 SOIL BIOLOGY 

4. Carbol-methylene blue: 

Methylene blue 1.5 grams 

Absolute alcohol 10 cc. 

Titrate in an evaporating dish and add 
gradually carbolic acid, 5 per cent aque- 
ous solution 100 cc. 

5. Eosin aqueous: 

Excellent for cell contents and cellulose walls. 

Eosin 1 gram 

Water 100 cc. 

6. Hsemalum : 

Hsematoxylin 1 gram 

Alcohol, 95 per cent, hot 50 cc. 

Then add to: 

Alum 50 grams 

Water, distilled 1000 cc. 

Cool, let settle, filter, and preserve from 
mold by vise of a crystal of thymol. 

Fixatives. 

1. Chromic acid, 1 per cent: 

Chromic acid 1 gram 

Water 100 cc. 

2. Osmic acid, 2 per cent : 

Osmic acid 2 cc. 

Water, distilled 100 cc. 

3. Picric acid: 

Picric acid 1 gi;am 

Water. 100 cc. 

Alcohol, 70 per cent 100 cc. 

4. Corrosive fixative: 

Corrosive sublimate, sat. solution 100 cc. 

Alcohol, 70 per cent 100 cc. 

For other information on stains, fixatives, and special 
methods consult texts on bacteriology, Plant Anatomy, 
by Stevens, Methods in Plant Histology, by Chamberlain, 
Behrens Tabellen, bei W. Behrens, and botanical literature. 



CHEMICAL METHODS. 

The chemical methods described in this manual have 
-been selected from the various possibilities in the different 
fields of chemistry, after thorough and painstaking research 
conducted under the conditions required in soil biological 
studies. 

QUANTITATIVE DETERMINATION OF NITROGEN. 

In all these methods determinations should be made in 
duplicate and blank determinations run on all reagents. 
All analyses are reported in milligrams per 100 grams of 
water-free soil, or air-dry soil, and as pounds per acre. 

Total Nitrogen in Soil. — The Kjeldahl method 
modified to include nitrateS is the most reliable method 
for the determination of total nitrogen in soils. 

Procedure. — Place 10 grams of soil (5 grams if an 
alkali or marine soil, 2 grams if a peat) in a 500 cc. Kjel- 
dahl flask, add 20 or 30 cc. sulfuric acid (according to 
organic matter content) containing 1 gram of salicylic 
acid, mix thoroughly and add 5 grams of sodium thiosul- 
fate, heat slowly at first; after 10 minutes boiling add 
1 drop of metalhc mercury (0.6 gram), continue digestion 
until the contents are grayish in color (about 2 hours), 
add potassium permanganate to a permanent pink color, 
transfer ,to a liter Kjeldahl flask (glass), using 250 cc. 
nitrogen-free distilled water. Place the receiving flask 
containing the standard acid in position and turn on the 
steam and air of the pipe distillation apparatus. Add the 
required alkali (60 cc.) containing the potassium sulfide to 
the Kjeldahl flask, connect with apparatus and distill at 
least 40 minutes, obtaining about 200 cc. of distillate. 
Titrate the distillate against ammonium hydroxid with 
sodium alizarin sulfonate or cochineal, as indicator. 

Ill 



112 SOIL BIOLOGY 

Total Organic Nitrogen in Soil. — Employ the 
method previously described, omitting the salicylic acid 
and sodium thiosulfate. This method is very reliable 
where nitrates are not a factor in soil studies. 

Total Nitrogen in Microorganisms, Plants, and 
Other Organic Materials. — The following method 
developed in this laboratory is a modification of the 
Kjeldahl-Gunning Arnold method and has given excellent 
success. It is rapid, accurate, and convenient. This 
method has been used constantly for determining nitrogen 
in green and dry plants and under these conditions" it is 
unexcelled. 

Procedure. — Place the sample (0.2-1 gram) in the 
500 cc. Kjeldahl, add 20 cc. sulfuric acid, about 6-8 grams 
of potassium bisulfate (fused) and mercury as in Kjeldahl 
method. Digest 1^-2 hours. Proceed to distill as in 
the Kjeldahl method, using eitlier pipe or tank distillation 
apparatus. The acid and alkali should be about A'^/20 
for accuracy. Sulfuric acid and sodium hydroxid are 
used when the amounts of nitrogen are small. If nitrates 
are a factor, apply the salicylic acid and sodium thio- 
sulfate modification and delay the addition of the mer- 
cury until reduction has proceeded 10-15 minutes. Titrate 
as usual. 

Ammonia Nitrogen. — The ammonia in soils, espe- 
cially in ammonification studies where large amounts are 
present, is most easily and satisfactorily determined by 
direct distillation with magnesium oxide. A slight hydroly- 
sis occurs, but aside from this the method is very reliable 
in the hands of students with the pipe distillation appara- 
tus used in this laboratory which has entirely eliminated 
the usual troubles. 

Procedure. — Place 100 grams (more may be used) of soil, 
either dry or moist, in a liter Kjeldahl flask, add 250 cc. 
water and 6-8 grams magnesium oxide. Place receiving 
flasks in position, turn on steam and air and connect 



CHEMICAL METHODS 113 

Kjeldahl flasks with apparatus, light burners, and distill 
45 minutes. Titrate as in total nitrogen method. 

Ammonia Nitrogen by Aeration. — The aeration 
method for the determination of ammonia is accurate 
and applicable to small amounts. The method herein 
described is a modification of the Folin method applied to 
soil and was developed in this laboratory. 

Procedure. — Place 50 grams of fresh soil (dry may be 
used) in a 500 cc. Kjeldahl flask, add 100 cc. water and 
5 grams of heated magnesium oxide, connect with a 400 cc. 
shaker bottle containing standard sulfuric acid (iV/20) . 
The apparatus is set up in battery of as many as 20 or 
more, and connected with a vacuum pump run by a motor. 
The air is washed with sodium hydroxid and sulfuric acid 
and drawn through the apparatus for 17-19 hours, or con- 
veniently over night. The titration is made against weak 
alkali (A7'/30), using rosolic acid indicator where small 
amounts of ammonia are present. 

Nitrite Nitrogen. — The determination of nitrite 
nitrogen in solutions is easily accomplished by the follow- 
ing method in which ammonia and nitrate do not interfere. 

Procedure. — ■ Filter the soil or the medium containing 
the nitrite into a 250 cc. Jena beaker and wash residue 2-3 
times with distilled water, add 50 cc. of dilute sulfuric acid 
(4 CC.-4000 water) very slowly, keep cool, and then add 
an excess of iV/10 potassium permanganate, let stand 5 
minutes, and then add 5 cc. of 10 per cent potassium iodide 
solution and titrate the free iodine with iV/10 sodium 
thiosulfate. (Hot starch solution may be used but is not 
necessary.) 1 cc. iV/10 permanganate = 0.7005 milli- 
grams of nitrogen. 

Nitrate Nitrogen. — The determination of nitrates 
alone is made by the Devarda method or the aluminum re- 
duction method. Ammonia and nitrites must be expelled. 

Procedure. — (1) The filtered solution or acid .extract 
of a soil is first made alkaline with nitrogen-free potassium 



114 SOIL BIOLOGY 

hydroxide and boiled until the ammonia is expelled, which 
requires about 20-30 minutes, depending upon the solution 
and the amount of ammonia present. Acidify with strong 
acetic acid, adding it frequently during evaporation. 
Evaporate to dryness on steam bath and take up with 
5 cc. acid and again run to dryness. The procedure in 
both methods is identical to this point. 

(2) In the Devarda method transfer to a 500 cc. 
Kjeldahl flask using 200 cc. water and add 0.5 gram De- 
varda metal and 4 cc. potassium hydroxid (300 grams per 
liter), distill one hour using heat, collecting in standard 
hydrochloric acid. Titrate as usual. 

(3) In the aluminum reduction method after running 
to dryness, the salts are transferred to a reduction tubs, 
50 cc. of water added, and 1-4 cc. nitrogen-free potassium 
hydroxid, a strip of aluminum, and reduction allowed to 
proceed over night. With solutions high in organic matter 
reduction is slow, and, the solutions should be tested for 
absence of nitrites or nitrates with diphenylamine sulfuric 
acid or a little Devarda metal should be added when dis- 
tillation is carried out. Distill 40 minutes and titrate as 
usual. Both these methods are accurate and convenient. 
The Devarda method is more rapid. The aluminum 
method is convenient where large numbers of analyses are 
to be made, and especially where only a limited amount 
of apparatus is available during a laboratory period. 

Nitrite and Nitrate Nitrogen. — The determina- 
tion of both nitrites and nitrates is made by either the 
Devarda or aluminum reduction methods. 

Procedure. — The solution should be made alkaline and 
the ammonia expelled; and then follows the procedure as 
outlined in the method for the determination of nitrate 
nitrogen under 2 or 3. 

Inorganic Nitrogen. — The determination of total 
mineral nitrogen is made by direct reduction and dis- 
tillation with Devarda metal or by reducing in the cold 



CHEMICAL METHODS 115 

and then distilling with the aluminum, taking care in the 
latter method to make the water in the trap acid, as 
ammonia may be given off if present in large amounts. 

Qualitative Tests for Nitrogen. 

. Organic Nitrogen. — Ignite a small portion of the 
substance on platinum foil and test the residue for inor- 
ganic salts. Test some of the original substance for 
nitrate. 

Prepare a stock solution by placing a piece of clean 
metallic sodium (sodium is kept in kerosene; remove and 
clean so no vapors of the oil will be mistaken for the 
sodium vapors) the size of a pea in a small 2-inch test 
tube. Place test tube in an iron clamp attached to an 
iron stand, add a little material and heat until the vapors 
of sodium form a layer | inch high. Place 3 drops of the 
liquid, or if a solid an equivalent amount, letting it fall at 
intervals of one to two seconds upon the sodium vapors, 
taking care not to let the substance touch the side walls. 
Quickly add a second piece of sodium and ignite strongly. 
By means of a pair of forceps carefully lower the hot tube 
into 10 cc. of distilled water in a beaker. Warm, filter, 
and use this stock solution for the tests of nitrogen and 
sulfur. It may also be used to test for chlorine, bro- 
mine, and iodine, if desired. If ammonia is a factor, add 
strong alkali and note the odor of ammonia. Organic am- 
monium salts give the nitrogen test and odor with alkali. 

Procedure. — Boil a few cc. of the alkaline stock solu- 
tion for two minutes with five drops FeS04 solution and 
one drop of FeCls solution. Cool, acidify carefully with 
HCl. If the precipitate does not disappear, leaving a blue 
or bluish green precipitate or a clear yellow solution, warm 
gently. Cool, and filter through a clean white filter paper 
and wash. A blue precipitate of Prussian blue shows the 
presence of nitrogen. Often, if iodine is present, a blue 
coloration may appear at this point. To distinguish from 



116 SOIL BIOLOGY 

the nitrogen test, wash the filter with alcohol to dissolve 
out the iodine. 

Test for Organic Nitrogen and Sulfur when 
Present Together. — Acidify one cc. of the stock solu- 
tion with HNO3 and add a drop of ferric chloride. A deep 
red coloration is due to the formation of ferric sulfo- 
cyanide. Always carry on tests for organic nitrogen and 
organic sulfur together with this test. 

Ammonia. — Place a few drops of the solution to be 
tested in a test tube or a Nessler tube, add 10 cc. or 50 cc. 
of water to mark on tube, and then add 0.1 cc. Nessler 
reagent. Yellowish coloration indicates ammonia. Test is 
accurate to 0.00025 of a part per million. Large amounts 
of ammonia are best shown by the ammonium chloride 
test. Whenever possible distill a portion of the solution 
to be tested. 

Nitrites. — The value of this qualitative test depends 
upon its use in the presence of nitrates and ammonia. 
The Greiss method is well suited to demonstrate the 
presence of nitrite. It is a delicate test and cannot be 
relied upon to indicate quantity unless performed as below. 

Place 0.2 cc. of the solution to be tested in 10 cc. of 
water, another 0.2 cc. in 20 cc. of water in test tubes, add 
0.5 cc. sulfanilic acid, and then 0.5 cc. of alpha naphthyl- 
amine acetate freshly made up. Note the number of 
minutes required at room temperature (22°-24° C.) for the 
color to appear as faint, medium, and strong. Compare 
with the table found below : 





Time, 
min. 


Color 


1 mg. of N 

1 

1 

3 


as nitrite from 50 cc 


solution in 10 cc 

10 cc 

20 cc 

10 cc 


11-2 

5 

5 

1 

3 

3 

5 

12 
i-i 


faint 

medium 

faint 


3 " 


10 cc 


strong 


3 


" 20 cc. ... 


3 
3 


20 cc 

" 20 cc 


medium 
strong 
strong 
strong 


5 " 


" 10 CO. . . 


10 


10 cc 



CHEMICAL METHODS 117 

A standard solution will be available for comparison. 
Always run blanks on water and reagents. 

Nitrates. — The most useful test for nitrates where 
nitrites are present is the paratoluidine sulfate reagent. 
Nitrites do not interfere permanently and 100 parts per 
million of nitrogen as nitrite may be present and not cause 
error on the nitrate determination. There m.ust be at 
least 80 parts per million of nitrogen as nitrate to give a 
reliable test. This test serves to show nitrates in sufficient 
amounts to be relied upon for making transfers and to 
determine when quantitative analysis should be attempted. 

Place 1 cc. of solution to be tested in a test tube, add an 
equal volume of concentrated sulfuric acid without mixing 
the liquids; then add 4 drops of the reagent paratoluidine 
sulfate solution. The test should stand 3-5 minutes. A 
red ring at the point of contact indicates the presence of 
nitrates. Nitrites give a yellowish brown coloration.. 
There is present at least 4 milligrams in 50 cc. of solution 
when a good red ring develops after 3 minutes. Brucine 
sulfuric acid is a delicate and excellent reagent for nitrates, 
giving a red coloration which later becomes yellowish red. 
Diphenylamine sulfuric acid gives an excellent blue colora- 
tion with the nitrate, while the nitrite color is brownish. 

Quantitative Determination of Sulfur. 

Total Sulfur in Soil and Organic Materials. — 
The total sulfur content of a soil or crops is determined by 
the bomb combustion method. 

Procedure. — The materials are first finely ground. 
Place 5 grams of soil, or 1 gram organic material, in the 
bomb cup and add 12 grams sodium peroxide for the soil, 
3 grams for the organic niatter, 1 gram magnesium, powder, 
thoroughly mix using caution not to scatter the sodium per- 
oxide. Place the cover on the bomb cup and make tight 
with the lock nut. Heat until bottom of bomb is red. 
Cool in water. Transfer fusion to beaker using warm 



118 SOIL BIOLOGY 

water, acidify with concentrated hydrochloric acid (34 cc. 
for 12 grams peroxide), adding 10 cc. in excess of neutrahty. 
Evaporate to dryness on steam bath; take up with 1-1 
HCl and again evaporate to dryness a second time. Take 
up with hot water and filter hot. The filtrate and wash- 
ings are heated to boiling and 15 cc. of a ten per cent solu- 
tion of barium chloride added with constant stirring. 
Place on steam bath and keep warm six hours. Filter 
and wash free of chlorides, dry and burn to constant weight, 
adding sulfuric acid after the first weighing and again burn 
to expel excess acid. Weigh as barium sulfate. 7.04 
mgs. of BaS04 — 1 mg. S. 

Another more convenient and a rapid method is that of ' 
the sulfur photometer. With the sulfur in solution ready 
for precipitation proceed as below. 

Make slightly acid with hydrochloric and make up to 
250 cc. in a graduated flask. Mix and take out 10 cc. to 
which 90 cc. of water is added. Place the 100 cc. in an 
Erlenmeyer flask, add 0.3-0.5 gram of barium oxalate 
powder (equal parts barium chloride and oxalic acid), cork 
immediately, and shake occasionally for 20 minutes. 
Adjust the graduated tube in the water in the crystallizing 
dish so the rounded end is under water (| inch of water). 
Use the electric fight.* Darken the room with the black 
shades. Place some of the solution in the separatory 
funnel, admit solution in tube until the last tip of the cone 
of the light just disappears. Remove tube, read in milli- 
meters the depth of liquid. Repeat reading three times, 
using same solution. Refer to the chart and report as 
milligrams of solution per 100 grams of soil and as pounds 
per acre. This method is accurate to 0.2 or 1 per cent 
and gives excellent results for sulfur in soils. Read the 
standard solution and report it with the results obtained. 
Sulfates in Soils. — Place 100 grams of soil in a 
400 cc. shaker bottle, add 200 cc. of water acidified with 
hydrochloric, 5 cc. per 1000, and shake for 7 hours in the 



CHEMICAL METHODS 119 

mechanical shaker, filter and make up to 250 cc. and pro- 
ceed as in the total sulfur determination, using sulfur 
photometer. 

Qualitative Test for Sulfur. 

Organic Sulfur. — To 1 cc. of the stock solution pre- 
pared for organic nitrogen test, add acetic acid and then 
lead acetate. A black precipitate indicates sulfur. 

Inorganic Sulfate. — Add barium chloride to the 
solution. A white finely divided . crystalline precipitate 
indicates sulfate. 

Hydrogen Sulfide. — For the gas use moistened lead 
acetate paper; add lead acetate in acetic acid solution. 

Determination of Phosphorus, Carbon, Dry Matter, 
Acidity, and Magnesium. 

Total phosphorus, carbon (total organic and inorganic), 
and magnesium in soils. Organic materials and raw rock 
phosphates. 

The methods given in "Soil Fertility Laboratory 
Manual," by Hopkins and Pettit, are used for these deter- 
minations. 

Determination of Calcium. 

Proceed as in the manual referred to above with the 
exception of titrating the oxalate against N/10 potassium 
permanganate instead of weighing as CaO. 1 cc. N/10 
KMn04 = 2 mgs. Ca. 

Determination of Iron. 

Total Iron. — The total iron is determined by reduc- 
ing all the iron present to the ferrous iron and then oxidiz- 
ing to ferric iron by A^/10 permanganate. 

Procedure. — Place 20 cc. of the solution to be analyzed 
in a 250 cc. beaker, add 50 cc. water and 15 cc. concen- 
trated sulfuric acid. Add zinc dust and heat if neces- 



120 SOIL BIOLOGY 

sary. Test a drop for complete reduction by placing it on 
the porcelain plate in contact with ammonium thiocyanate. 
Red coloration indicates ferric iron. If no ferric iron is 
present cool and titrate with N/10 potassium perman- 
ganate. 1 cc. iV/10 KMn04 = 0.0056 Fe, = 0.0072 
FeO, = 0.0080 FeaOs. 

Carbon Dioxid. — The carbon dioxid evolved during 
decomposition of organic residues is determined by collect- 
ing it in standard potassium hydroxid solution contained 
in fermentation valves. Remove the valve, wash the 
contents with 200 cc. hot water into an Erlenmeyer flask, 
add 3-5 cc. of a ten per cent solution of neutral barium 
chloride, shake, let stand a few minutes and titrate the 
excess potassium hydroxid with standard acid preferably 
of equivalent strength. 1 cc. N/2 KOH = 11 mgs. CO2. 



MECHANICAL METHODS. 

Collecting Soil Samples for Biochemical Analy- 
sis. — The collection of soil samples for biochemical 
studies should be carried out in the same manner as sam- 
pling for soil analysis which consists of taking 16-20 
borings per tenth acre plot, by means of the soil auger 
or soil tube. This ensures a representative sample. 
Duplicate samples are taken to a depth of 6| inches, 20 
inches, and 40 inches as desired. The borings are mixed 
and the required amount placed in a properly labeled 
Mason fruit jar. This makes the work comparable and 
meets the practical requirements. 

Collecting Soil Samples for Bacteriological 
Analysis. — A different problem is presented in sampling 
a given area for bacteriological analysis than that of 
sampling for soil analysis. The irregular results obtained 
from samples which include the surface two inches have 
caused them to be discarded in taking the sample. It has 
also been found that the soil auger is responsible for con- 
tamination, especially when the subsurface and subsoil 
are sampled. 

The sampling is accomplished by removing the surface 
two inches with a sterile spatula and then with a sterile 
spatula dl*awing the sample. Place it on a sterile oilcloth 
or in a sterile soil pan, mix, and place the composite sample 
in a sterile container. This does not disturb the soil 
materially, and suffices for surface samples. A special 
soil tube which is constructed of brass, pointed at one end^ 
and which is plugged with cotton at the other end, has 
recently been recommended. Such a tube made longer 
than the one proposed by Noyes (Jour. Am. Soc. Agron. 
(1915) 6, 239) should prove valuable for obtaining samples 

121 



122 SOIL BIOLOGY 

deeper than 6f inches. For studying the bacterial flora 
at greater depths than the surface 6f inches the pit method 
has been employed. 

It consists in digging a pit to the required depth to 
which it is desired to obtain the last sample. The sides 
of the pit are cut down with a spade as sharply as the soil 
will permit, and then they are sterilized either by direct 
flaming or scraped with a sterile spatula. The samples 
are then removed from the sides at right angles to the 
vertical axis. This may be done with a sterile soil tube 
or a sterile spatula. The sample is treated as in the other 
method outlined from this point. 

The container used may be either a sterile cotton- 
plugged bottle or a sterile fruit jar. The spatulas and 
other apparatus are sterihzed by flaming in the field. (An 
alcohol lamp is used.) A sterile agate pan is a suitable 
substitute for the oilcloth. The samples should not be 
taken when the wind is blowing over a light breeze unless 
the soil is moist on the surface. Early in the morning, 
6-9 o'clock, is usually the best time for taking samples. 

The error due to contamination of the sample in the 
field is very slight if the above conditions are fulfilled. 
The soil samples are used in the desired experiments 
immediately after collection. 

Preparation of the Soil Samples. — Soil samples for 
biochemical analysis of field experiments are not dried for 
the determination of ammonia. For the moisture and 
nitrate determinations the samples are dried in an electric 
oven at 108° C. for 8 hours. Samples collected for bac- 
teriological analysis are immediately used for inoculation 
or incubation. 

Samples intended for laboratory practices (to be used 
from December to April) are air-dried; sieve to remove 
stones and large roots and place in barrels or soil bins, for 
use later. Some soils are pulverized, but in no case are 
they ground as in soil analysis. Dry soil is satisfactory 



MECHANICAL METHODS 123 

for demonstrating the principles involved in most soil 
biological studies. A supply of fresh, moist soil is always 
maintained in the greenhouse which serves any time as a 
source of fresh inoculating material. 

Sampling of Crops. — The use of common farm 
materials instead of artificial materials, such as casein, 
blood meal, etc., is taken as the standard in these studies. 
Samples of hays, straws, stover, and such like should be 
collected in the field so that their history may be accu- 
rately known. Decomposition is related to the stage of 
development of crops and it is, therefore, of advantage to 
know the condition of sample. Samples should be air- 
dried and ground if used in beaker experiments. Grind- 
ing to pass a 2-mm. or 10-mesh sieve is sufficient. A card 
catalogue of all soil samples, limestones, phosphates, and 
crop samples is a great convenience. The analysis is 
entered on the card together with the other necessary 
data. Farm and green manures are collected and used 
whenever possible. 

Shaking. — A mechanical shaker is convenient for use 
in preparing soil infusions and soil extracts. 

Preparing a Soil Infusion. — The bacteria are satis- 
factorily removed from the soil particles by shaking the 
soil with water. Place 100 grams of soil in a 400 cc. 
sterile shaker bottle and add 200 cc. sterile distilled water. 
Place a clean rubber in the bottle and shake for 5 minutes. 
Allow solution to settle 15 minutes and then use as desired 
by means- of sterile pipettes. 

Ignition of Soil. — Ignited soil is used in culture solu- 
tions, especially for nitrite and nitrate bacteria. Place 
the soil in an iron or nickel crucible and burn at a red 
heat until all the organic matter is completely oxidized. 
Large quantities may be burned in a kiln and stored for use. 

Centrifuge. — A rapid separation of bacteria and soil 
can be made by use of the centrifuge. It is also valuable 
in obtaining extracts containing enzymes and the Uke. 



124 SOIL BIOLOGY 

Filtration. — ' Soil solutions are filtered for chemical 
determinations and for making soil extract media. A 
battery of suction filters is of great advantage in obtaining 
clear soil extracts. A rapid filtration can be made through 
glass wool or absorbent cotton. Most soil biological media 
are filtered through cotton. The Berkefeld and Pasteur- 
Chamberland filters are indispensable for obtaining bac- 
teria free filtrates. 

Cleaning Glassware. — It is absolutely necessary 
that all glassware shall be perfectly clean. Acids, alka- 
lies, and organic matter do not permit equal distribution of 
the solutions to be examined. 

The test tubes, Petri dishes, and flasks are cleaned in 
the following way: Boil in soap and water for 10-15 
minutes or immerse in the following hot cleaning solution 
and leave over night: 

Potassium bichromate 60 parts 

Concentrated sulfuric acid 460 parts 

Water 300 parts 

(Add acid slowly with constant stirring.) 

Wash with water, rinse with distilled water, and invert 
to dry. Test tubes may be easily dried in the hot-air 
oven. 

The tumblers are washed in the ordinary way with tap 
water, rinsed with distilled water, and inverted to dry. 

It is well to plug the test tubes and flasks with cotton 
when clean and dry. (See the instructor about rolling 
plugs.) 

For most glassware which is used for chemical work 
simple washing in tap water with a cleanser, then with 
distifled water and finally rinsing with distilled water is 
sufficient. Dipping in weak hydrochloric acid and then 
rinsing in distilled water is efficient for a great deal of the 
glassware employed in the determination of nitrogen. 

Cleaning cover-slips and slides requires especial atten- 
tion since the success of flagella staining and obtaining 



MECHANICAL METHODS 



125 



good permanent preparations depends to a great extent 
upon clean slips and slides. The following method has 
given best results in this laboratory. Wash in distilled 
water, boil 10 minutes in strong nitric acid, remove and 
wash in distilled water (do not handle the slips or slides 
with your hands or dirty forceps), and then wipe dry. 
Place in absolute alcohol in containers used only for this 
purpose. Test the cover-slips and slides with distilled 
water to see if they are clean by dj-awing the film over the 
surface at will. 

If alkali is used caution must be exercised as hot alkali 
or strong alkali dissolves the glass, producing an etched 
appearance. 

Autoclave. — The autoclave represents sterilization 
by moist heat under pressure. It is by far the most satis- 
factory means of sterilization for most media, solutions, 
and materials that will stand heating under pressure. 

The autoclaves 4B and 2B are connected with high- 
pressure steam. To operate, proceed as follows: Open 
cocks under doors and lower cock under autoclave. Turn 
on slowly the high pressure steam cock (upper cock under 
autoclave), one-fourth turn at first, increasing gradually 
later. 

The table below is a guide to the use of the autoclaves. 



Material 


Pressure, 
pounds 


Time. 


Water 


12 
12 
15 
15 
10 
15 
15 


10 min. . 












30 min. 


Urea, dextrose 

Sand 


15 min. 


Soil 


6-8 hours 







In sterilizing soil, wait until all the air is expelled from 
it t)efore closing the autoclave. For such masses use the 
maximum thermometers in the interior of the mass during 
sterilization. 



126 



SOIL BIOLOGY 



TABLE OF AUTOCLAVE — TEMPERATURES AND 
PRESSURES. 





Steam pressure, 
pounds 


Temperature. 




C. 


F. 





100 

109 

115.5 

118.0 

121.5 

126.5 

131 

134.5 


212 


5 


228 


10 


240 


12 


244 


15 


250 


20 


260 


25 


268 


30 


274 







Hot-air Oven. — The hot-air sterilizer has four com- 
partments. The burners are in the lower compartments. 
To operate turn on the gas by pulling the levers at either 
end. Light burners and close doors. When the ther- 
mometer in the door registers 350° F. (20-25 minutes), 
pull the levers at each end shut. Usually the oven is 
loaded with all the glassware to be needed for a long time 
or with duplicate sets and allowed to run 5-8 hours. 

Sterilization of Glassware. — Invert the clean 
Petri dishes and place them in the round seamless tin 
boxes* assigned for this purpose. Place the cover on the 
box and sterilize by heating in the hot-air oven at least 1 
hour at 350° F. The boxes are inverted before opening 
to prevent possible contamination of the Petri dishes. 

Pipettes are handled as follows: Plug the mouth end 
with cotton and then place them in the horizontal copper 
boxes used in sterilizing them. Heat in the hot-air oven 
as above. It is not always necessary to plug the pipettes 
if commonplace caution is exercised in their use after 
sterilizing. 

Tumblers, cylinders, and other glassware which cannot 

* A 24-ounce round seamless tin box used in the manner above 
has been foimd to give excellent satisfaction. The boxes should be 
heated several hours before using the first time, owing to their being 
lacquered. 



MECHANICAL METHODS 127 

be safely subjected to dry heat or steam sterilization can 
be effectively sterilized by letting them stand in (1-300) 
solution mercuric chloride for 20 minutes and rinsing with 
sterile water. 

Glass bottles, lipless Jena glass beakers, or tile pots can 
be used in place of tumblers and permit of sterilization in 
the autoclave. 

Sterilization of Seeds. ' — It has been found extremely 
difficult to completely sterilize seeds for use in experiments 
where sterile conditions are required in the beginning or 
throughout. A satisfactory method is wanting for all 
kinds of seeds. However, in a great deal of the work, com- 
plete sterilization is not necessary, especially is this true of 
legume studies. To recognize the possibilities and elimi- 
nate the necessity of complete sterilization will often spell 
success in these experiments. 

A solution of mercuric chloride (1-500) for 10 minutes 
gives an efficient sterilization against legume, non-symbi- 
otic nitrogen fixers, nitrite, nitrate, ammonifiers, and many 
other organisms but does not insure kiUing mold spores. 

Solutions of mercuric chloride, hydrogen peroxide, cop- 
per sulphate, bromine, silver nitrate, and suKuric acid 
sometimes give rather unsatisfactory results owing to the 
persistence of air bubbles on and inside the seeds. 

Hutchinson and Miller used a solution of mercuric 
chloride in a vacuum apparatus. This obviated the 
trouble from air bubbles. 

Treatment with a 5 per cent solution of chloride of fime 
for three hours has given good results at this laboratory. 

Harrison and Barlow suggest a method which is suited 
to special investigations. Pods are picked from plants 
while they are yet green. They are then washed in 1-1000 
mercuric chloride for one hour and dried in folds of sterile 
cotton. The pods are then burned by holding in a flame 
with sterile forceps, after which they are opened and the 
seeds placed in folds of sterile cotton. When dry they 



128 SOIL BIOLOGY 

are removed to plugged sterile test tubes by means of 
sterile forceps. 

Large seeds with tough seed-coats are taken in forceps, 
dipped in alcohol (95 per cent) and passed through a low 
flame. Cowpeas and soybeans have been successfully 
treated in this way in this laboratory. 

Sterilization of Nodules. — The nodules are effec- 
tively sterilized by treatment with 1-500 mercuric chloride 
for 3 minutes, washing in sterile water, and then crushing 
in another portion of sterile water in a sterile container 
with sterile glass rod. 

Sterilization of Parts of Plants. — It is sometimes 
necessary to sterilize the stem or leaf of a plant for inocu- 
lation purposes. It has been found that 1-1000 mercuric 
chloride rubbed into the leaf or stem will not only give very 
satisfactory sterilization but seems to penetrate the tissue 
and tends to keep the cells sterile. In inoculating experi- 
ments this fact should be considered as it may destroy the 
inoculation. For further methods consult Irwin Smith's 
"Bacteria in Relation to Plant Diseases, I." 

Sterilization of Soil. — It is often necessary to 
sterilize a soil for experimental purposes. This subject 
has been studied by many workers and the present 
methods are included below. 

It is important for the student of soil biology to note 
the changes which have been found to occur in the steriliza- 
tion of a soil, as they account for the irregular behavior 
observed in many experiments. It will be recalled that 
sterile conditions have been stated as being very unsatis- 
factory for plant growth. Some of the reasons are to be 
found in the following paragraphs. 

The three common methods are: 

1. Moist heat (autoclave). 

2. Dry heat (hot-air oven). 

3. Volatile antiseptics. 



MECHANICAL METHODS 129 

Moist Heat — The autoclave is the only satisfactory- 
means of attaining complete sterilization aside from fire. 
The changes which occur increase with a rise in tem- 
perature. 

The chemical changes brought about by steam under 
pressure in the soil are many and complex, but only a few 
are of importance. These are the nitrates, nitrites, am- 
monia, and precipitation reactions, such as formation of 
insoluble calcium, phosphorus, and iron compounds. 

Lyon and Bizzell concluded that 30 pounds pressure for 
two or four hours reduced the soil nitrates and nitrites to 
ammonia. They also found a great increase in the am- 
monia content after sterilization. This ammonia origi- 
nates chiefly from organic matter. Schreiner and Lathrop 
found a notable increase in many forms of organic nitrogen, 
an increase in water soluble constituent and in acidity 
upon treating at 30 pounds pressure for three hours. 
Thus it is seen that steam sterilization greatly increases 
the soluble matter of a soil and changes the organic matter 
more than either of the other methods. 

The biological effects are evidenced by complete death 
of all forms of life in the soil and the deleterious influence 
exerted on plant growth for 2-3 months after treatment. 
Some investigators have noted very great beneficial 
results to plant growth after sterilization by this method, 
planting, however, after the soil has been weU aerated. 
Sometimes this increase amounts to 4-10 times the crop 
obtained on the unsterilized soil. 

The physical characters suffer in a similar way as in 
the dry heat method which is described below. 

In order to determine correctly the temperature of the 
interior of the mass of soil a maximum thermometer should 
be inserted into the soil. 

Dry Heat. — The use of dry heat for soil steriUzation 
changes the chemical, biological, and physical properties 
of the soil. 



130 SOIL BIOLOGY 

The chemical studies have shown that the nitrogen 
undergoes changes. The total nitrogen may or may not 
remain constant according to its state of decomposition 
or according to the type of soil. Some soils yield ammonia 
and volatile organic nitrogenous decomposition products 
when heated much above 110° C. 

The soluble nitrogen is greatly increased by the heating 
due to changes brought about in the insoluble forms, and 
the amount made soluble in this way is related to the 
moisture content of the soil. 

Heating at 200° C. changes the organic matter of soils. 
At this temperature the soluble material is increased from 
6 to 10 times. 

Heating at 100° C. and 250° C. increases the solubility 
of all the mineral constituents except sodium in both water 
and fifth normal nitric acid as solvents. At 100° C, there 
is an increase in water-soluble calcium, magnesium, phos- 
phorus, sulfur, and bicarbonates. Potassium, silicon, and 
aluminum increased in half the soils tested while iron de- 
creased in most instances. 

Heating at 250° C. or ignition produced similar results. 
At 150° C. nitrates decomposed while at 200-250° C, 
practically total destruction of nitrates took place. Am- 
monia was produced in large amounts at 200° C, At 
200° C, 25 per cent of the total nitrogen was lost. 

The life of the soil is rendered almost extinct for the 
time being when a soil is heated at 95° C, or above. 
This temperature kills the vegetative stages of most of the 
plant life and the active stages of animal life. The spores 
are not destroyed completely until ignition is reached. 
The fauna and flora of any soil may be changed at any 
temperature above 45-50° C. Russell and Hutchinson 
found from two to four times the crop on a soil heated at 
95° C. as on an unheated soil. Many results indicate 
that plants grow better on heated than unheated soils. 

Physically the differences appear to be in the increased 



MECHANICAL METHODS 131 

capillary and absorptive power of the soil as determined 
by Richter in 1896, when he heated a soil to 100° C. for 
six hours on three consecutive days. 

Volatile Antiseptics. — This method is being studied 
with great interest at the present time in many laboratories. 
At best, it is only partial sterilization acting very similar 
to dry heat at 98° C. 

Carbon bisulphide and toluene are used most commonly. 
Chloroform, ether, xylol, and others may be employed 
for partial sterilization. Four per cent of toluene is 
effective. 

This method of treatment kills the living organisms but 
does not injure the spores or the encyst forms of life. The 
life of the soil greatly increases after this treatment and 
plant growth is more vigorous than on untreated soils. 
The increased chemical results noted are due to biological 
changes. Ammonia and nitrate production are greatly 
increased at first. Unless kept under sterile conditions, 
these differences gradually subside. Air-drying a soil 
rapidly or soils which have been dried a long time give 
similar results when the soil is again placed under normal 
conditions. This is due to a suppression of the fauna and 
flora during drying. The rate of multiplication of the 
bacteria under such conditions makes them the predomi- 
nant form o life and the consequent yield of ammonia and 
nitrate is high at first, but later, when the soil remains 
under normal conditions, the numbers and activities fall 
to the same level as fresh or untreated soil. 

Sterilization of Sand. — No permanent changes are 
caused by sterilizing sand. It should be well aerated after 
sterilization before being used as a medium for plants. 
The ignited soil is unchanged by sterilization. 



132 SOIL BIOLOGY 

POT-CULTURE METHODS. 

The value of data obtained in pot-.culture experiments 
is recognized by the soil biologist. Under the controlled 
conditions afforded in the well-equipped greenhouse many 
facts, unsolvable under field conditions, are established. 

The following paragraphs are included to assist the 
student in considering the essentials for successful pot- 
culture experiments. 

Containers. — The one-gallon earthen jar, one-gallon 
battery jar and the four-gallon earthen jars are satisfac- 
tory containers. A galvanized pot of the Wagner type is 
excellent as the water may be added beneath the surface. 
Evidence of zinc poisoning has been obtained in experi- 
ments of long duration with zinc pots. For accurate 
studies glass jars are preferable although the cost and break- 
age are high. The one-gallon earthen jar has a capacity 
of 10 pounds of soil and 12 pounds of sand. Drainage 
is provided by placing a glass tube in the bottom. 

Sand Medium. — Crystal white sand is an excellent 
medium for studying the elements of plant food. It 
should be washed free of salts with hydrochloric acid and 
with distilled water until free of acid. When great ac- 
curacy is required other methods such as ignition and 
reduction are necessary. 

Soil Medium. — Soil should be used as the medium 
ultimately and whenever it will not vitiate the results. 
The previous history is desirable. It should be sieved 
and mixed carefully before being placed in the container. 
Clay soils are more difficult to experiment with owing to 
their preventing drainage and root development. Car- 
bonates will greatly decrease this trouble. 

Moisture. — The optimum moisture content should 
be established and maintained throughout accurate experi- 
ments. This in many cases is determined by an empirical 
method. Weighing the jars at 4-7 day intervals and 



POT-CULTURE METHODS 133 

adding water in equal amounts during these periods to all 
treatments has proved very satisfactory. 

Plant Food. — The plant food elements are con- 
veniently applied in solutions as given in "' Soil Fertility 
Laboratory Manual," Hopkins and Pettit. Calcium car- 
bonate and dolomite are applied as the dry salt, 10 grams 
per gallon jar. 

Inoculation of Legume Seeds. — Excellent results 
are obtained when inoculating sand and soil cultures by 
the following method. Wash the nodules to be used for 
inoculation with mercuric chloride 1-300 for 10-12 
minutes; then wash in sterile water and finally crush in 
another portion of sterile water. This avoids the trans- 
ference of an unnecessary number of organisms not con- 
cerned in the legume experiment. Place a few cubic 
centimeters (5 cc. per seed if large or with small seeds 
10-20 cc. per jar) of this infusion on each seed before 
covering with sand or soil. This method is the simplest 
and best for pot-culture experiments. A few nodules are 
sufficient to make several liters. It is not necessary to 
sterilize with the mercuric chloride solution in all cases. 
This method of inoculation has never failed to give excel- 
lent results at this station. Pure cultures from the 
laboratory or a soil infusion may be used with good 
results. Where added soil will not affect the experiment, 
it may be employed for inoculation. The glue method 
may also be used. 

Planting. — A careful selection of seeds to be planted 
is important. Irregular seeds and those with ruptured 
coats should be discarded. In many experiments the seeds 
should be accurately weighed in order to have a check on 
their chemical composition. A high per cent of germina- 
tion is desirable. Usually it is advisable to plant a few 
more seeds than necessary for the final stand so that they 
may be thinned to a definite number per jar which number 
depends upon the size of the jar and the kind of plant. 



134 SOIL BIOLOGY 

Crops. — Plants differ in their adaptability to grow 
under greenhouse conditions and only experience can 
make fine distinctions as to the best choice of crop. As 
a rule, annuals are more uniform in their growth than 
biennials or perennials; this is especially true of the 
legumes. Cowpeas, soybeans, and the cereal crops are 
well suited to these methods. 

In the selection of the crop, the temperature should be 
considered as well as the light values. The fall is a poor 
time for the growing of most crops as the light is not 
intense enough. Warm- weather crops will stand the tem- 
perature of the greenhouse even in summer. Sudden 
changes in temperature should be avoided as they are 
disastrous. 

Plant diseases and insects are a serious menace to 
greenhouse experiments and a careful operator should be 
employed to regulate the temperature and fumigate the 
house properly. Distilled water sprayed on the plants 
checks the ravages of red spiders. Sulphur may be found 
useful as a temporary remedy for mildews. An interesting 
case of contamination came under the writer's observation 
in which red spiders carried the cowpea organisms from 
one jar to another until all became inoculated. 

Growth of Plants under Sterile Conditions. — 
This is not a desirable method of experimentation owing 
to the abnormal conditions which it involves. It is, how- 
ever, sometimes necessary to grow plants under sterile 
conditions in order to establish the influence of a definite 
factor or to determine a specific reaction. Bell jars, 
WouLfe bottles, beakers, or battery jars covered with 
cotton will be found of service in this connection. A side 
tube jar is often valuable for such studies. Erlenmeyer 
flasks and test tubes can be used by plugging with 
cotton. Various devices have been tried with partial 
success. 

Records. ■■ — Accurate records must be kept of all the 



POT-CULTURE METHODS 135 

facts of importance, such as the history of the soil, plant 
food* applied, moisture content, kind of seed, number of 
seeds planted and the number allowed to remain, irregu- 
larities in growth, injuries due to insects, or from other 
causes, and of greatest importance, the weights or yields. 

Photographs should be taken as their worth may prove 
inestimable. Measurements of growth are often valuable 
data. The harvested materials are usually air-dried or 
oven-dried and placed in properly labeled receptacles for 
storage until ready for analysis. 



SUGGESTIONS FOR INSTRUCTORS AND 
STUDENTS PREPARING TO TEACH 

ACID, ALKALI, AND OTHER STANDARD 
SOLUTIONS. 

The number of standard solutions required in a course 
of this kind is necessarily greater than in courses dealing 
with but a single field of chemistry or biology. 

Experience has demonstrated the value of the solutions 
herein found: 

- Solution. Use. 

1. iV/20 NaOH Titrating media and for use in titrating 

small amounts of nitrogen. 

2. N/20 HCl Titrating media. Not used very much 

as most solutions are at the correct 
reaction. 

3. iV/1 NaOH Correcting reaction. 

4. N/l HCl Correcting reaction. 

5. N/KNO3 Acidity determinations. 

6. N/7 HCl Nitrogen determination when amount 

is sufficient to give more than a dif- 
ference of 2 cc. in titration. 

7. N/7 NH4OH Used as above. 

8. Ar/20 H2SO4 Titrating small amounts of nitrogen. 

9. iV/1483 KOH Det. P. 1 cc. = 0.2 mg. phosphorus. 

10. iV/1483 HNO3 1 cc. = 0.2 mg. phosphorus. 

11. N/IO KMn04 1 cc. = 2 mg. Ca, calcivun nitrite. 

1 cc. = 0.7005 mg. nitrite, iron deter- 
mination. 

Nitrite and iron determinations. 

Phosphorus determination. 

Culture media. 

Culture media. 

Culture media. 

Nitrite determination. 
136 



12. AT/lONazSaOs.... 

13. 6 per cent FeCls. . 

14. 10 per cent FeCla. 

15. 10 per cent NaCl. 

16. 10 per cent CaCU 

17. 10 per cent KI. . . 



ACID, ALKALI, AND OTHER SOLUTIONS 137 

18. Reduced KOH (300 

grams per liter) 

solution Nitrite and nitrate determinations. 

19. Ammonium molyb- 

date solution .... Phosphorus determination. 

20. Saturated ammo- 

nimn oxalate solu- 
tion Calcium determination. 

21. Nessler solution ... . Ammonia determination. 

22. NaOH (2 pounds per 

hter (26.6 grams 

K2S) Nitrogen determinations. 

INDICATORS. 

The following indicators have been selected from a 
large number tested as being most reliable. 

FormuloB of Indicator. Alkali and Acid Color and Use, 

1. Sodium alizarin sulfonate, 1 

gram AlkaUes red — acids yellow. 

Alcohol, 60 per cent, 100 cc. . . In all nitrogen determinations 

where ammonia is distilled. 

Best indicator where H2S is not (See 6. RosoUc acid.) 
present. Volatile organic 
bases do not affect this in- 
dicator as much as the 
others. 

2. Cochineal, 3 grams AlkaUes violet — acid yellow- 

ish-red. 
Macerate in the solution 
below: 

Water, 200 cc In nitrogen determinations 

Alcphol, 60 cc. where sulfides or sulfur dis- 

tUl over in amoimts suffi- 
cient to obscm-e the end 
point of number 1. 
This indicator is only .slightly 

affected by HjS or CO2. 

3. Lacmoid, 3 grams Alkalies blue — -acids red. 

Water, 700 cc Alternative for Number 1. 

" Alcohol, 300 cc. 
This indicator is affected by 
HoS. 



138 SOIL BIOLOGY 

4. Methyl orange, 1 gram Alkalies yellow — acids red. 

Water, 1000 cc Strong acids. 

Not affected by H2S or CO2 . . . Sodium silicate, hydrochloric 

acid, sodium carbonate. 

5. Phenolphthalein, 1 gram Alkali red — acid "colorless. 

Alcohol, 50 per cent, 100 cc. . Phosphorus, media, acidity. 
Very sensitive to CO2 Valuable for weak alkalies,* 

carbonates, alkali earths. 

Useless for NH4S Bicarbonates neutral to this 

indicator, organic acids. 

6. Rosohc acid (commercial), 1 Alkali rose red — acid yel- 

gram low. 

Alcohol, 60 per cent, 100 cc . . . Ammonia titration when 

aluminum or copper are used 
Useless for acetic acid. Not for reduction. Use sodium 
affected by ammonia but by hydroxid and sulfuric acid 
ammoniima salts. as large amounts of am- 

monium salts interfere. 

COLORIMETRIC REAGENTS. 

Soil solutions and other turbid solutions which retain 
their color upon filtering through the ordinary filters may be 
decolorized by use of aluminum cream (alkali-free). Two 
to three cc. per liter usually suffices; repeat if necessary. 
It is best, however, to avoid as much as possible the use 
of such materials on account of occlusion. 

FormulcB of Reagents. Use and Color. 

1. Diphenylamine sulfuric acid. . . . Nitrite — brownish blue. 

Diphenylamine, 1 gram Nitrates — blue. 

Sulfuric acid (cone), 100 cc Rings on a palate. 

Small amount of ferric salts 

do not interfere Delicate test. 

2. Brucine suKuric acid Red ring shows nitrates, 

Brucine, 1 gram Delicate test. , 

Sulfuric acid (cone), 100 cc. 

3. Paratoluidine sulfate Red ring with nitrates. 

Paratoluidine, 0.5 gram Not delicate. 

Sulfuric acid (cone), 100 cc. 

Very well adapted to indicat- 
ing nitrates when more than 
2-3 mgs. of nitrogen present 
in 25 cc. of solution. 



CHEMICALS USED BY STUDENTS 139 

4. Alpha naphthylamine acetate Nitrites. Very delicate. See 

and sulfuric acid qualitative test for nitrites, 

page 116. 

(a) Alpha naphthylamine, 1 
gram. 

Acetic acid, sp. gr. 1.04, 200 
cc. 

(b) Sulfanilic acid, 1 gram. 
Acetic acid, sp. gr. 1.04, 250 cc. 

Use equal amounts of each 
when making a test. The . 
alphanaphthylamine should 
be made up fresh each 2-3 
days. 

A list of chemicals and apparatus have been included as 
a guide and represent the materials which have been found 
essential with the present state of development of the 
course. For special work a larger number of sugars, 
organic acids, organic salts, stains, and special chemicals 
than found in the list included are necessary. A greater 
variety of chemicals and apparatus are necessary in a 
course of this kind owing to the varied nature of the 
experiments. 

CHEMICALS USED BY STUDENTS IN SOIL 
BIOLOGY. 



Acid, acetic. 


Agar agar shredded. 




' carbolic. 


" " powdered. 




' chromic. 


Alcohol, ethyl. 




' citric. 


Alpha naphthylamine. 




' hydrochloric. 


Aluminum metal strips. 




' molybdic. 


" metal powder (coarse) 




' nitric. 


" metal powder (fine). 




' oxalic. 


" and potassium sul- 




' osmic. 


fate. 




' picric. 


Ammonium carbonate. 




' salicylic. 


hydrate, sp. gr. 0.90 




' sulfanilic. 


" nitrate. 




' sulfuric. 


" oxalate. 



140 



SOIL BIOLOGY 



Ammonium diphosphate. 

" monophosphate. 
" sulfate. 
" thiocyanate. 
Anilin oil. 
Asparagin. 
Balsam, Canada. 
Barium chloride. 
Barium oxalate powder. 
Barium hydroxid. 
Blood meal. 
Beef extract. 
Brucine. 

Calcium carbonate (ground lime- 
stone) . 

" acetate. 

" chloride. 

" nitrate. 

" oxide. 

" triphosphate. 

" diphosphate. 

" monophosphate. 

" sulfate. 
Calcium sulfate (gypsum). 
Calcium sulfate (plaster of 

Paris) . 
Carbon bisulfide. 
Casein. 
Chloroform. 
Cleanser. 
Collodion. 
Copper suUate. 
Dextrin. 
Dextrose. 
Diphenylamine. 
Dolomite. 
Ether. 
Feldspar. 

Ferric ammonium citrate. 
" wire. 
" chloride. 
Ferrous sulfate. 
Formaldehyde. 



Gelatin. 

Glass wool. 

Glycerin. 

Glue (furnitiu-e). 

Hydrogen peroxide. 

Iodine. 

KaoUn. 

Kieselguhr. 

Lacmoid. 

Lead acetate. 

" oxide. 
Lysol. 

Magnesium ammonium phos- 
phate. 

" carbonate. 

" chloride. 

" metal 

" oxide. 

" sulfate. 

Maltose. 

Manganese sulfate. 
Mannite. 
Mercury metal. 
Mercuric chloride. 
Metal, Devarda's alloy. 
Oil, immersion cedar. 
Oil, cedarwood. 
Oil, cylinder, heavy, light. 
OU, cloves. 
Oil, paraffin. 
Faratoluidine. 
Paraffin, M. P., 52° C. 
Paraffin, M. P., 40-42° C. 
Paraffin, M. P., 50-53° C. 
Peptone, Witte's. 
Potassium bichromate. 
Potassium bisulfate (fused). 
Potassium carbonate. 
Potassium chloride. 
Potassium hydroxid. 
Potassium iodide. 
Potassium nitrate. 
Potassium permanganate. 



APPARATUS 



141 



Potassium phosphate, mono- 
basic. 
Potassium phosphate, dibasic. 
Potassium sulfate. 
Potassium sulfide. 
Rock phosphate. 
Saccharose. 
Silver nitrate. 
Silver nitrite. 
Sodiimi asparaginate. 

carbonate. 

chloride. 

hydroxid. 

(metal) . 

nitrate. 

nitrite. 

peroxide. 

sUicate. 

sulfate. 

sulfide. 
Sodium thiosulfate. 
Stains, Bismarck brown. 

Eosin. 

Fuchsin. 

Gentian violet. 



Hsematein. 

Hsematoxylin. 

Methyl blue. 

Methylene blue. 

Nigrosin 

Orange G. 

Safranin O. 

Versuvin. 
Indicators : 

Cochineal. 

Congo red. 

Litmus. 

Methyl orange. 

Phenolphthalein . 

Sodium ahzarin sulfo- 
nate. 

Starch, potato. 
" corn. 
Toluene. 
Urea. 
Vaseline. 
Xylol (xylene). 
Zinc, granulated. 

" dust. 
Wood ashes. 



APPARATUS. 



Asbestos. 

Balance, triple beam. 

" metric solution scale. 
" weighing scoops. 
Beakers, various sizes. 
Bottles, various sizes. 
Boxes, copper for pipettes. 

" seamless tin for Petri 
dishes. 

" sUde. 
Brushes, test tube, flasks. 
Bulbs, distilling, Hopkins. 
Bui-ettes, various sizes. 
Burners, Bunsen, pilot, and 

micro. 



Casserole, 210 cc. 

Cell, blood counting chamber. 

Clamps, test tube, burette. 

Corks. 

Cotton. 

Crucibles, porcelain, Gooch, iron. 

Cylinders, graduated, various 

sizes. 
Dishes, evaporating. 

" Petri, dia. larger dish 
100 mm. 

" depth of lower dish 16 
mm. 
Files. 
Filter cases for 6 sizes. 



142 



SOIL BIOLOGY 



Flasks, Erlenmeyer, boiling, filter. 
" Kjeldahl (500, 1000 cc). 
" volumetric and KoUe. 
" various sizes. 
Forceps, cornet, dissecting. 
Funnels, glass, Buchner, copper. 
Glasses, jelly, with tin covers. 
Glass rods, tubing. 
Jars, anatomical, staining, speci- 
men. 
Lamp, alcohol. 
Lenses, magnifying, reading 

glass. 
Lens paper. 

Microscopic slides 3X1, concave 
3X1. 
" cover glasses, round, 

square, several 
thicknesses. 
Motors, porcelain, agate. 
Paper, litmus. 

" filter, 6 sizes. 
Pencils, red, blue for glass. 
Pipettes, 1, 2, 5, 10, 25, 50, 100 
cc. 
" graduated. 
" stopcock, 10 cc. 
Plates, porcelain, 12 cavities. 



Plates, Lafars counting. 

Platinum foil, wire 0.41 mm. di- 
ameter. 

Pump filter. 

Rings, iron. 

Rubber stoppers, policemen, 
tubing. 

Section lifters. 

Shears, steel. 

Sieves, brass nested, set of 5. 

Spatulas, 3 sizes. 

Spoons, bone. 

Stopcocks, Geissler. 

Supports, iron, funnel test tube. 

Test tubes, 150 mm. long out- 
side. 

Thermometers, several kinds. 

Towels, barber. 

Tongs. 

Triangles. 

Tripods. 

Tubes, fermentation (Smith's), 
fermentation safety valve 
in top. 

Watch glass. 

Wire baskets. 

Wire gauze. 



SPECIAL APPARATUS. 

Aeration apparatus, battery of Colorimeter, Nessler tube, double 

16 ' mirrors. 

Autoclave, 2B, 4B (large). Crocks, earthen, for waste ma- 
Auger, soil, 1, IJ inch. terial. 

Balance, analytical, No. 10 and Digestion racks. 

weights. Distillation apparatus, battery of 
Balance, analytical. No. 16 and 20. 

weights. Filters, Berkefeld. 
Baths, steam. " Pasteur Chamberland. 

Centrifuge and accessories (elec- Incubators, 37°, 20°, room tem,' 

trie). perature. 



SPECIAL" APPARATUS 



143 



Jars, dialyzers. 

" sample. 
Microscopes, regular and binoc- 

iilar, and accessories. 
Microtome. 

Oven, electric, drying and incu- 
bator (small). 

" gas, hot-air sterilizer 
(large). 
Pans, soil. 



Plate, electric, hot. 
Photometer sulfur. 
Pump, vacuum for aeration ap- 
paratus. 
Shaking machine. 
Stools, laboratory. 
Trays, laboratory, various sizes. 
Tables, laboratory. 
Tube, soil. King. 



